WO2023056971A1

WO2023056971A1 - Anti-cd47 antibody formulation

Info

Publication number: WO2023056971A1
Application number: PCT/CN2022/124085
Authority: WO
Inventors: Zhenping Wu; Karen TWU; Chongdong FU; Feng Wang; Yinxing SHEN; Shanhua ZHONG; Jiazhou WU
Original assignee: Hutchmed Limited
Priority date: 2021-10-09
Filing date: 2022-10-09
Publication date: 2023-04-13
Also published as: TW202330027A; AR127270A1

Abstract

Provided is an anti-CD47 antibody formulation, a preparation method therefore and the use thereof.

Description

ANTI-CD47 ANTIBODY FORMULATION

Technical Field

The present invention relates to an anti-CD47 antibody formulation, a preparation method therefor and the use thereof.

Background

CD47 (Cluster of Differentiation 47) was firstly identified as the tumor antigen of human ovarian cancer in 1980s. CD47, also known as an integrin-associated protein (IAP) , ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multiple membrane receptor belonging to an immunoglobulin superfamily that has a single immunoglobulin-like domain and five membrane spanning regions. As a ligand of signal regulatory protein α (SIRPα) , CD47 binds to the V-like domain at the NH ₂ terminus of SIRPα. SIRPα is expressed primarily on bone marrow cells, including macrophages, granulocytes, dendritic cells (DCs) , mast cells and their precursors, e.g. hematopoietic stem cells. CD47 on normal cells binds to SIRPαon macrophages, which releases the “don’ t eat me” signal, and thereby inhibits the phagocytic function of macrophages. It is an important mechanism how macrophages distinguish self from non-self in the innate immune system. CD47 is widely expressed on human tumor cells and tissues, including acute myelogenous leukemia (AML) , chronic granulocytic leukemia, acute lymphocytic leukemia (ALL) , non-Hodgkin’s lymphoma (NHL) , multiple myeloma (MM) , bladder cancer and other solid tumors. The tumor cells escape from the phagocytosis of macrophages though the binding of highly expressed CD47 to SIRPα on the surface of macrophages, which favors tumor growth. The immune checkpoint CD47 is considered to be a target which is potentially effective and can be widely used for tumor immunotherapy. At present, a variety of specific blockers have been developed to target the CD47/SIRPα interaction. There are a number of preclinical and clinical trials being carried out, which relates to the drugs including anti-CD47 antibodies and SIRPα fusion proteins for treatment in diffuse large B cell lymphoma, acute myelogenous leukemia, and advanced solid tumors. These drugs are administered either alone or in combination with other anti-tumor drugs. Taking the anti-CD47 antibody Hu5F9 developed by Forty Seven company as an example, in a phase I clinical trial to evaluate the effect of Hu5F9 in the treatment of 22 patients with lymphoma, the combination of Hu5F9 and Rituximab could produce objective remission in 50%of patients who did not respond to Rituximab alone. According to the clinical data published in 2019, the complete remission rate of 14 patients with recurrent/refractory acute myelogenous leukemia treated with Hu5F9 in combination with Azacitidine is up to 36%, and the remission rate of 11 patients with bone marrow suppression syndrome treated with Hu5F9 in combination with Azacitidine is up to 55%.

As with any protein therapeutic agent, antibodies are subject to physical and chemical instability factors such as aggregation, denaturation, crosslinking, deamidation, isomerization, oxidation and clipping. When an antibody at a high concentration is comprised in the formulation, it is more difficult to retain the physical stability and chemical stability of the antibody. Therefore, it is of significant importance to determine the optimal formulation conditions to improve the antibody stability during production, storage and usage.

Therefore, the present invention provides a pharmaceutical formulation comprising a high concentration of an anti-CD47 antibody or an antigen-binding fragment thereof; in particular, the antibody or the antigen-binding fragment thereof has a high anti-tumor activity and does not induce a significant red blood cell agglutination reaction, thereby satisfying more clinical requirements; and the pharmaceutical formulation comprising the antibody or the antigen-binding fragment thereof exhibits a good stability under multiple simulated physical stress conditions that potentially occur during drug production or transportation.

Summary of the Invention

The present invention provides a pharmaceutical formulation containing an anti-CD47 antibody or an antigen-binding fragment thereof, especially a stable liquid formulation with a high concentration of the antibody.

In one aspect, the present invention provides a pharmaceutical formulation with a pH of about 5.0 to about 8.5, or about 5.5 to about 7.0, comprising (i) an anti-CD47 antibody or an antigen-binding fragment thereof; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

In some embodiments, the buffer in the pharmaceutical formulation of the present invention is a histidine salt buffer or a citrate buffer. In some embodiments, the histidine salt buffer is histidine-histidine hydrochloride buffer. In some embodiments, the citrate buffer is citric acid -disodium citrate buffer.

In some embodiments, the buffer is at a concentration of about 10 mM to about 80 mM, preferably about 20 mM to about 50 mM, such as about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, or about 70 mM.

In some embodiments, the stabilizer in the pharmaceutical formulation of the present invention is at least one selected from the group consisting of a sugar, an amino acid, a polyol.

In some embodiments, the stabilizer comprises at least one amino acid, such as arginine, preferably arginine hydrochloride.

In some embodiments, the stabilizer consists of an amino acid, preferably arginine hydrochloride.

In some embodiments, the sugar preferably is sucrose.

In some embodiments, the stabilizer consists of an amino acid and a sugar, preferably arginine hydrochloride and sucrose.

In some embodiments, the amino acid is at a concentration of about 1 mg/mL to about 100 mg/mL, such as about 5 mg/mL to about 40 mg/mL.

In some embodiments, the sugar is at a concentration of about 10 mg/mL to about 100 mg/mL, preferably about 20 mg/mL to about 80 mg/mL.

In some embodiments, the ratio of mass concentration of the amino acid to the sugar is about 1 : 1 to about 1 : 15, such as about 1 : 1, about 1 : 2, about 1 : 3, about 1 : 4, about 1 : 5, about 1 : 6, about 1 : 7, about 1 : 8, about 1 : 9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 : 13, about 1 : 14, or about 1 : 15.

In some embodiments, the surfactant in the pharmaceutical formulation of the present invention is selected from polysorbate surfactants, preferably polysorbate-80 or polysorbate-20.

In some embodiments, the surfactant is at a concentration of about 0.002% (w/v) to about 0.5% (w/v) , preferably about 0.01% (w/v) to about 0.05% (w/v) , such as about 0.01% (w/v) , about 0.02% (w/v) , or about 0.03% (w/v) , preferably about 0.02% (w/v) .

In some embodiments, the anti-CD47 antibody or the antigen-binding fragment thereof in the pharmaceutical formulation of the present invention is at a concentration of about 10-150 mg/mL, preferably about 40-120 mg/mL, such as about 20 mg/mL, about 50 mg/mL, about 60 mg/mL, about 80 mg/mL, about 100 mg/mL, or about 110 mg/mL.

In some embodiments, the pharmaceutical formulation of the present invention has a pH of about 5.5 to about 7.5, about 5.5 to about 7.0, such as about 6.0 to about 7.0, preferably about 6.0 to about 6.5, more preferably about 6.5.

In one aspect, the present invention provides a pharmaceutical formulation, comprising:

(i) about 10-150 mg/mL of the anti-CD47 antibody or the antigen-binding fragment thereof;

(ii) about 10-80 mM of the citrate buffer or the histidine salt buffer;

(iii) about 5-40 mg/mL of arginine hydrochloride and about 0-80 mg/mL of sucrose; and

(iv) about 0.01% (w/v) to about 0.05% (w/v) of polysorbate-80,

with a pH of about 6.0-7.0.

(i) about 40-120 mg/mL of the anti-CD47 antibody or the antigen-binding fragment thereof;

(ii) about 20-80 mM of the citrate buffer or the histidine salt buffer;

(iii) about 5-40 mg/mL of arginine hydrochloride and about 20-80 mg/mL of sucrose, wherein the ratio of mass concentration of arginine hydrochloride to sucrose is about 1 : 1 to about 1 : 15, and

(iv) about 0.01% (w/v) to about 0.05% (w/v) of polysorbate-80, with a pH of about 6.0-7.0.

(ii) about 20-80 mM of the citrate buffer;

(iii) about 5-40 mg/mL of arginine hydrochloride, and

In some embodiments, the pharmaceutical formulation of the present invention is in a liquid form, which further comprises a vehicle, including but not limited to water, such as water for injection, bacteriostatic water for injection (BWFI) , double distilled water or a combination of at least two of them.

In one aspect, the present invention also provides a lyophilized pharmaceutical formulation, which is prepared, by means of lyophilization, from the pharmaceutical formulation of the present invention described above.

In one aspect, the present invention also provides a reconstituted pharmaceutical formulation, which is prepared by the reconstitution of lyophilized pharmaceutical formulation of the present invention with a reconstitution medium.

In some embodiments, the reconstitution medium, including but not limited to at least one selected from the group consisting of water for injection (WFI) , bacteriostatic water for injection (BWFI) , a sodium chloride solution (such as 0.9% (w/v) NaCl) , and a glucose solution (such as 5% (w/v) glucose) .

In some embodiments, the reconstituted pharmaceutical formulation of the present invention further comprises a vehicle, including but not limited to water, such as water for injection, bacteriostatic water for injection (BWFI) , double distilled water or a combination of at least two of them.

In some embodiments, the pharmaceutical formulation of the present invention (including the liquid pharmaceutical formulation , lyophilized pharmaceutical formulation or reconstituted pharmaceutical formulation) can be a formulation in an injectable or infusible liquid form.

In one aspect, the present invention also provides an article of manufacture, comprising a container filled with the pharmaceutical formulation (including the liquid pharmaceutical formulation, lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) of the present invention.

The anti-CD47 antibody or the antigen-binding fragment thereof suitable for the pharmaceutical formulation of the present invention can be any anti-CD47 antibody or an antigen-binding fragment thereof.

In one aspect, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from HCDR1, HCDR2 and HCDR3 of a heavy chain variable region (VH) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7.

In one aspect, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21.

In one aspect, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from LCDR1, LCDR2 and LCDR3 of a light chain variable region (VL) , wherein the VL comprises the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In one aspect, the anti-CD47 antibody or antigen-binding fragment thereof comprises one to three selected from light chain complementary determining region 1 (LCDR1) , LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3, wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises three CDRs of a heavy chain variable region (VH) , i.e., HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL) , i.e., LCDR1, LCDR2 and LCDR3; wherein the VH and VL are selected from:

(1) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 2;

(2) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 3, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(3) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 5, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10;

(4) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 9 or 10; or

(5) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.

In some embodiments, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprises heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, and light chain complementary determining region 1 (LCDR1) , LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21, the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, and light chain complementary determining region 1 (LCDR1) , LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 17, the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) , wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7, and the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH and VL are selected from

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 1, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 2.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 3, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 4.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 5, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 6, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 6, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 8.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 9.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 10.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is, for example, a human IgG1 constant region, a human IgG4 constant region, a human IgG4P constant region or a human IgG1TM constant region. The human IgG4P constant region of the present invention is a mutant human IgG4, which has an amino acid substitution of S228P (numbered according to EU) . In some embodiments, the human IgG1TM constant region is a mutant human IgG1 constant region, which has amino acid substitutions of L234F, L235E and P331S (numbered according to EU) .

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain constant region, such as a human κ or λ constant region.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody. In some embodiments, at least part of the framework sequence of the anti-CD47 antibody or antigen-binding fragment thereof is a human consensus framework sequence. In one embodiment, the anti-CD47 antibody or antigen-binding fragment thereof is a full-length antibody, a single-domain antibody such as a VHH, a Fab, a Fab’ , a Fab’ -SH, a (Fab’ ) ₂, a single-chain antibody such as a scFv, a Fv, a dAb (domain antibody) or a bis (multi) -specific antibody.

In another aspect, the anti-CD47 antibody or antigen-binding fragment thereof in the pharmaceutical formulation of the present invention can be replaced with an immunoconjugate or immune fusion comprising the anti-CD47 antibody or antigen-binding fragment thereof described herein.

In some embodiments, the pharmaceutical formulation is an aqueous pharmaceutical formulation. It is contemplated that the concentration of the antibody present in the formulation is determined by, for example, a desired dose volume and administration mode. The pharmaceutical formulation of the present invention contains a high concentration of the anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is at a concentration of about 10 mg/mL to about 150 mg/mL, such as about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, or about 150 mg/mL, including all values and ranges therebetween. In some embodiments, the antibody is at a concentration of about 50 mg/mL or about 80 mg/mL.

In one aspect, the present invention also provides the use of the pharmaceutical formulation (including the liquid pharmaceutical formulation, lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) described herein for preventing or treating CD47-related diseases in a subject.

In another aspect, the present invention provides a method of treating or preventing CD47-related diseases in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical formulation (including the liquid pharmaceutical formulation, the lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) described herein.

In another aspect, the present invention also provides the use of the pharmaceutical formulation (including the the liquid pharmaceutical formulation, lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) described herein for preventing or treating CD47-related diseases in a subject.

In another aspect, the present invention also provides the use of the anti-CD47 antibody or antigen-binding fragment thereof for the preparation of the pharmaceutical formulation described herein, wherein the pharmaceutical formulation (including the the liquid pharmaceutical formulation, lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) is used for preventing or treating CD47-related diseases in a subject.

In some embodiments, the CD47-related diseases include hematological cancer and solid tumor, including but not limited to bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcomas, glioma, glioblastoma, myeloma, endometrial cancer, renal carcinoma, (benign) melanoma, prostate cancer, thyroid carcinoma, cervical cancer, gastric cancer, liver cancer.

In another aspect, the present invention also provides the use of the pharmaceutical formulation (including the lyophilized pharmaceutical formulation or the reconstituted pharmaceutical formulation) described herein in the preparation of drugs for treating or preventing CD47-related diseases.

In some embodiments, the CD47-related diseases are preferably cancers, for example, the cancers comprise hematological cancer and solid tumor, such as bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcomas, glioma, glioblastoma, myeloma, endometrial cancer, renal carcinoma, (benign) melanoma, prostate cancer, thyroid carcinoma, cervical cancer, gastric cancer, liver cancer, colon cancer, ovarian cancer, urothelial carcinoma, and so on.

The pharmaceutical formulationof the present invention can also be combined with other therapeutic agents or therapeutic modes, for treating or preventing CD47-related diseases.

The present invention also encompasses any combinations of any embodiments described herein. Any embodiments or any combinations thereof described herein are applicable to any and all anti-CD47 antibodies or fragments, methods and uses thereof of the present invention described herein.

Brief Description of the Drawings

Figure 1 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Raji cells.

Figure 2 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Toledo cells.

Figure 3 shows the binding affinity of antibody HMA02h14-48 to CD47 on the surface of REC-1 cells.

Figure 4 shows the activity of antibody HMA02h14-48 in blocking the interaction between human CD47 and SIRPα.

Figure 5 shows the effect of antibody HMA02h14-48 on phagocytosis of Raji cells by human MΦ.

Figure 6 shows the effect of antibody HMA02h14-48 on phagocytosis of Toledo cells by human MΦ.

Figure 7 shows the effect of antibody HMA02h14-48 on phagocytosis of REC-1 cells by human MΦ.

Figure 8 shows the effect of antibody HMA02h14-48 on phagocytosis of HL-60 cells by human MΦ.

Figure 9 shows the effect of antibody HMA02h14-48 on agglutination of the red blood cell in vitro.

Figure 10 shows the ability of antibody HMA02h14-48 to bind to CD47 on the surface of human red blood cells.

Figure 11 shows the inhibition of Toledo tumor growth by Hu5F9 and HMA02h14-48.

Figure 12 shows the inhibition of REC-1 tumor growth by Hu5F9 and HMA02h14-48.

Detailed Description of the Invention

Definitions

Unless otherwise stated, the present invention will be implemented using conventional techniques in molecular biology (including recombinant techniques) , microbiology, cell biology, biochemistry and immunology, which are within the technical scope in the art.

In order that the invention may be more readily understood, some of the scientific and technical terms are defined as follows. Unless otherwise explicitly defined elsewhere herein, all scientific and technical terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. With respect to definitions and terms in the art, reference can be made to Current Protocols in Molecular Biology (Ausubel) by professionals. The abbreviation of an amino acid residue is a standard 3-letter and/or 1-letter code used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes the corresponding plural form thereof, unless otherwise explicitly specified.

The term “about” means a value or composition within an acceptable error range of the particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” can refer to a scope within 1 or more than 1 standard deviations according to practices in the art. Alternatively, “about” can refer to a range of up to 5%, 10%or 20% (i.e., ± 5%, ± 10%or ± 20%) .

When used to connect two or more optional items, the term “and/or” should be understood to mean any one of the optional items or any two or more of the optional items.

As used herein, the term “comprise” or “include” means to include the mentioned elements, integers, or steps, but does not exclude any other elements, integers, or steps. As used herein, when the term “comprise” or “include” is used, unless otherwise indicated, it also encompasses instances composed of the mentioned elements, integers or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to encompass an antibody variable region composed of the specific sequence.

The terms “integrin-associated protein (IAP) ” or “CD47” , when used herein, refers to any natural CD47 from any vertebrate source, including mammals (such as primates (e.g., humans) and rodents (e.g., mice and rats) ) , unless otherwise stated. The term covers a “full length” unprocessed CD47 and any form of CD47 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD47, such as splice variants or allelic variants. In some embodiments, CD47 refers to a full length CD47 or fragment thereof (such as a mature fragment thereof lacking a signal peptide) from a human. In some embodiments, a human CD47 refers to CD47 identical to the amino acid sequence as set forth in NCBI accession number NP_001768.1 or a fragment thereof. In some embodiments, the term also covers a fusion protein comprising CD47 or a fragment thereof.

“SIRPα” means a wild-type signal regulatory protein α, or recombinant or non-recombinant polypeptide comprising amino acid sequence of wild-type signal regulatory protein α, or a natural or naturally occurring allelic variant of signal regulatory protein α.

The term “affinity” as used herein refers to the strength of the sum total of all the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless otherwise indicated, as used herein, “binding affinity” refers to an intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen) . The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K _D) . Examples of analyses known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., ForteBio) .

The term “CD47-related disease” as used herein refers to a non-physiological state related to the expression or function or activity of CD47, or to the activity of the signal transduction mediated by CD47, including but not limited to cancers. In some embodiments, the diseases will benefit from the blocking of a CD47-related signal transduction.

The term “signal transduction” as used herein refers to a biochemical causal relationship generally initiated by protein-protein interactions such as the binding of CD47 to its receptor, which relationship leads to the transmission of signals from one part of the cell to another part of the cell. Generally, transmission includes the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that cause the signal transduction. The penultimate process generally includes nuclear events, thereby causing changes in gene expression.

The terms “activity” and “biological activity” or the terms “biological property” and “biological feature” as used herein are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize CD47 activity in vivo or in vitro, enhancement or activation of CD47 activity, IC ₅₀, in vivo stability of an antibody and immunogenicity of an antibody. Other identifiable biological properties or features of antibodies known in the art include, for example, cross reactivity (i.e., cross reactivity, generally with non-human homologs of targeted peptides, or with other proteins or tissues) , and the ability to maintain high levels of antibody expression in mammalian cells. The properties or features mentioned above can be observed, determined or evaluated using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasmon resonance assay, in vitro or in vivo neutralization assay, receptor binding, production and/or secretion of cytokines or growth factors, signal transduction and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other sources) .

The term “antibody” as used herein refers to any form of antibody having a desirable bioactivity. Therefore, it is used in the broadest sense, including but not limited to a monoclonal antibody (including a full-length monoclonal antibody) , a polyclonal antibody, a multispecific antibody (such as a bispecific antibody) , a humanized antibody, a human antibody, a chimeric antibody, a CrossMab antibody, or a camelized single-domain antibody.

The terms “whole antibody” , “full-length antibody” , and “intact antibody” are used interchangeably herein and refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH region and VL region can be further divided into a hypervariable region (being a complementary determining region (CDR) ) , among which a more conservative region (being a framework region (FR) ) is interspersed. A “complementary determining region” or “CDR region” or “CDR” is a region in an antibody variable domain, which is hypervariable in sequence and forms a structurally established loop ( “hypervariable loop” ) and/or contains an antigen contact residue (“antigen contact point” ) . CDR is mainly responsible for binding to antigen epitopes. CDRs of heavy chain and light chain are generally called CDR1, CDR2 and CDR3, which are numbered sequentially from the N-terminus. The CDRs located in an antibody heavy chain variable domain are called HCDR1, HCDR2 and HCDR3 respectively, while the CDRs located in the antibody light chain variable domain are called LCDR1, LCDR2 and LCDR3 respectively. Each VH or VL consists of three CDRs and 4 FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The constant region is not directly involved in the binding of an antibody to an antigen, but shows multiple effector functions.

In a given VH or VL amino acid sequence, the accurate amino acid sequence boundary of each CDR can be determined by using any one of the various well known schemes or a combination thereof, including, for example: Chothia scheme (Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins” , Journal of Molecular Biology, 196, 901-917 (1987) ) ; Kabat scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) and Contact (University College London) ; North scheme (North et al., A New Clustering of Antibody CDR Loop Conformations” , Journal of Molecular Biology, 406, 228-256 (2011) ) . The boundary of the CDR of the anti-CD47 antibody in the present invention can be determined according to any schemes or a combination thereof in the art and personal evaluation.

The light chain of the antibody can be classified into one of two types (referred to as kappa (κ) and lambda (λ) ) based on the amino acid sequence of the constant domain thereof. The heavy chain of the antibody can be mainly divided into 5 different types according to the amino acid sequence depending on the heavy chain constant region thereof: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

An “antibody in the form of IgG” refers to the IgG form of the heavy chain constant region of the antibody. For example, an antibody in the form of IgG4 means that the heavy chain constant region thereof is derived from IgG4.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the various antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations generally include a large number of antibodies being directed against different epitopes (or specific for different epitopes) . The modifier “monoclonal” indicates the feature of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring any particular method to produce the antibody.

The term “antigen-binding fragment” of an antibody as used herein includes fragments or derivatives of the antibody. Generally, the antigen-binding fragment includes at least one fragment (such as one or more CDRs) of the antigen-binding region or variable region of the antibody, and maintains at least some of the binding properties of the antibody. Examples of an antigen-binding fragment include, but are not limited to Fab, Fab', F (ab') ₂ and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., sc-Fv) ; and nanobodies and multispecific antibodies formed from antibody fragments. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragments or derivatives generally maintain at least 10%of the antigen-binding activity of the antibody from which they are derived. Preferably, the binding fragments or derivatives maintain at least 20%, 50%, 70%, 80%, 90%, 95%or 100%or more of the antigen binding activity of the antibody from which they are derived.

An epitope is a region of an antigen that is bound by an antibody. An epitope can be formed from contiguous amino acids or non-continuous amino acids juxtaposed by tertiary folding of a protein.

The term “chimeric antibody” as used herein refers to an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species. Generally, the variable domain is obtained from the antibody of the experimental animals such as rodents, while the constant domain sequence is obtained from an human antibody, so that the obtained chimeric antibody is less likely to induce adverse immune response in human subjects than the antibody from the experimental animals.

The term “humanized antibody” as used herein refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies. In general, the humanized antibody comprises at least one, and generally two, variable domains, in which all or substantially all of the hypervariable loops are correspond to those of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are correspond to those of human immunoglobulin. The humanized antibody can optionally comprise at least a portion of a constant region (Fc) derived from a human immunoglobulin. In some cases, as is known to a person skilled in the art, amino acid mutations can be introduced into humanized antibodies (e.g., variable domains, framework regions, and/or constant regions (if present) ) , for example, to improve certain properties of the antibodies; such antibody forms still fall within the scope of the “humanized antibody” of the present invention.

As is known to a person skilled in the art, the antibody may have a sugar chain of the cell for producing the antibody. For example, when produced in mice, in mouse cells, or in hybridomas derived from mouse cells, the antibody may contain a mouse sugar chain. Alternatively, when produced in rats, in rat cells, or in hybridomas derived from rat cells, the antibody may contain a rat sugar chain.

The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. The term includes native sequence Fc region and variant Fc regions. The native sequence Fc region covers a variety of naturally occurring immunoglobulin Fc sequences, such as various Ig subtypes and allogeneic Fc regions thereof (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu. 2014.00520. ) . In one embodiment, the Fc region of the human IgG heavy chain extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the lysine at the C-terminus (Lys447) of Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in Fc region or constant region is numbered in accordance with the EU numbering system, also referred to EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The terms “Fc region variant” or “variant Fc region” as used herein are used interchangeably herein, and mean that a modified Fc region is comprised relative to an Fc region of native sequence. The Fc region variants of the present invention are defined according to the amino acids modifications to the amino acids that compose them.

As used herein, an “immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels. An “immune fusion” is an antibody which is fused by covalently linking to one or more other peptides or polypeptides.

The term “pharmaceutical formulation” refers to a preparation which is in such a form as to permit the biological activity of the active ingredient (s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered and generally comprising or consisting of an active ingredient and a pharmaceutically acceptable excipient. Preferably, Such formulations are sterile.

The term “anti-CD47 antibody formulation” means a preparation comprising an anti-CD47 antibody as an active ingredient and a pharmaceutically acceptable excipient. After combined with the excipient, the anti-CD47 antibody as an active ingredient is suitable for therapeutic or prophylactic administration to a human or non-human animal. The antibody formulation of the present invention can be prepared, for example, as an aqueous liquid formulation, e.g., in a ready-to-use pre-filled syringe, or as a lyophilized formulation which is reconstituted (i.e., redissolved) by dissolution and/or suspension in a physiologically acceptable solution immediately prior to use. In some embodiments, the anti-CD47 antibody formulation is in the form of a liquid formulation.

“Pharmaceutically acceptable excipients” are those that can be reasonably administered to a mammal subject to provide an effective dose of the ingredient employed. Suitable pharmaceutically acceptable excipients are well-known in the art and include but are not limited to a buffer, a stabilizer, a surfactant, and a vehicle.

A “stable” pharmaceutical formulation refers to the protein (such as an antibody) in the formulation that essentially retains an acceptable degree of its physical stability and/or chemical stability and/or biological activity after storage under specific conditions. A variety of analytical techniques are known in the art for measuring the stability of proteins, see, e.g., Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N. Y., Pubs (1991) and Jones, A., Adv. Drug Delivery Rev. 10: 29-90 (1993) . Stability can be measured at a selected temperature and for a selected storage time period. In one embodiment, the pharmaceutical formulation can be stable for at least 4 weeks at room temperature or at 40℃, and/or stable for at least 6 months, preferably stable for at least one year, and more preferably stable for at least two years at 2-8℃. In one embodiment, the pharmaceutical formulation is preferably stable following freezing (to, e.g., -70℃) and thawing.

If the formulation does not exhibit aggregation, precipitation, turbidity and/or denaturation, or exhibits very little aggregation, precipitation, turbidity, and/or denaturation after storage for a period of time, the antibody can be considered to “retain its physical stability” in the formulation. Sub-visible aggregates in the formulation can be detected by light scattering methods and soluble aggregates in the formulation can be detected by SEC-HPLC. In addition, the stability of the formulation can be indicated by visually inspecting the appearance and visible particles of the formulation, or by determining the turbidity of the formulation by the OD350 nm method, or by determining the purity of the formulation by the non-reduced CE-SDS method. An “acceptable degree” of physical stability can denote the change in the percentage of the antibody monomers in the formulation of no more than 10%after storage at a specific temperature for a specific period of time. In some embodiments, an acceptable degree of physical stability may manifest itself as no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%change in the percentage of the antibody monomers. The pharmaceutical formulation can also be considered stable if the change in the percentage of the antibody monomers is no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

If the antibody in the formulation does not exhibit significant chemical changes after storage for a period of time, the antibody can be considered to “retain its chemical stability” in the formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody. For example, charge variants of the antibody in the formulation can be detected by cation exchange chromatography (CEX) or imaged capillary isoelectric focusing electrophoresis (icIEF) . In one embodiment, the stability of the formulation is measuring by determining the change in the percentage of charge variants of the antibody in the formulation after storage at a specific temperature for a specific period of time, wherein the smaller the change, the higher the stability of the formulation. An “acceptable degree” of chemical stability can denote the change in the percentage of charge variants (e.g., principal component, acidic component or basic component) in the formulation of no more than 30%, e.g., 20%, after storage at a specific temperature for a specific period of time. In some embodiments, an acceptable degree of chemical stability may manifest itself as the change in the percentage of charge variants (as principal components) of no more than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The pharmaceutical formulation can also be considered stable if the change in the percentage of charge variants (acidic components) is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%.

If the antibody in the formulation does not exhibit significant changes in the biological activity after storage for a period of time, the antibody can be considered to “retain its biological activity” in the formulation. For example, the antibody/antigen binding can be detected by ELISA, FACS, or BIACORE, etc. An “acceptable degree” of biological activity denotes the change in biological activity percentage of the antibody in the formulation of no more than 30%, 20%, or 10%after storage at a specific temperature for a specific period of time.

The term “lyophilized formulation” refers to a composition obtained or obtainable by a freeze-drying process of a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

w/v refers to weight volume ratio. Unless otherwise indicated, for example, 0.01%(w/v) refers to 0.1 mg/mL.

The term “reconstituted” formulation refers to a liquid formulation obtained by dissolving and/or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution. The reconstitution can be carried out by using a reconstitution medium, including but not limited to water for injection, bacteriostatic water for injection (BWFI) , a sodium chloride solution (such as 0.9% (w/v) NaCl) , and a glucose solution (such as 5% (w/v) glucose) .

The term “isotonic” formulation means that the formulation of interest has substantially the same osmolality as human blood. Isotonic formulations will generally have an osmolality ranging from about 250 to 350 mOsm/kg. For example, isotonicity can be measured using a vapor pressure osmometer or ice-freezing osmometer.

As used herein, the term “vehicle” refers to a liquid which can be used to dissolve or suspend an active ingredient and an inactive ingredient to form a pharmaceutical formulation. The vehicle that can be used in the present invention includes but is not limited to water, such as water for injection, bacteriostatic water for injection (BWFI) , double distilled water or a combination thereof.

The terms “carcinoma” and “cancer” refer to or describe physiological disorders in mammals, generally characterized by unregulated cell growth. This definition includes benign and malignant cancers and resting tumors or micrometastasis.

In some embodiments according to the present invention, the “treatment” of a disease or condition refers to the improvement of the disease or condition (i.e., alleviating or preventing or reducing the progression of the disease or at least one of its clinical symptoms) . In some other embodiments, “treatment” refers to relieving or improving at least one body parameter, including those physical parameters that may not be discernible by the patient. In some other embodiments, “treatment” refers to the regulation of a disease or condition physically (e.g., stabilization of discernible symptoms) , physiologically (e.g., stabilization of body parameters) , or both. Methods for evaluating the treatment and/or prevention of a disease are generally known in the art unless explicitly described herein.

In yet other embodiments according to the present invention, “prevention” of a disease or condition includes inhibition of the occurrence or development of the disease or condition or the symptom of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term “prevention” refers to administration of drugs to a subject prior to the onset of conditions or symptoms of cancer, in particular, in a subject at risk of cancer.

In some embodiments, after “treating” the cancer by the method of the present invention, an individual patient is considered to have been successfully treated if the individual shows one or more of the following: the number of cancer cells was decreased or cancer cells disappeared completely; tumor size was decreased; infiltration of cancer cells into peripheral organs was inhibited or absent, including, for example, the spread of cancer cells to soft tissues and bones; tumor metastasis was inhibited or absent; tumor growth was inhibited or absent; one or more symptoms associated with the specific cancer were relieved; incidence and mortality were reduced; the quality of life was improved; the tumor incidence, frequency or tumorigenicity was reduced; the number or frequency of cancer stem cells in tumor was reduced; tumor cells were differentiated into a non-tumorigenic state; or a combination of some of the effects.

“Inhibition of tumor growth” refers to any mechanism by which tumor cell growth can be inhibited. In some embodiments, tumor cell growth is inhibited by delaying tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by stopping tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis. In some embodiments, tumor cell growth is inhibited by inducing tumor cell differentiation. In some embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

As used herein, “sequence identity” refers to the degree of identity of sequences based on one by one nucleotide or amino acid comparing in the comparison window. The “(percentage) sequence identity” can be calculated as follows: comparing the two optimally aligned sequences in the comparison window, determining the number of positions with the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size) , and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment for purposes of determining the percentage of sequence identity can be achieved in various ways known in the art, for example, using publicly available computer softwares such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art is able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences or the target sequence area being compared. In the present invention, for antibody sequences, the percentage of identity of amino acid sequences is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and then performing an optimal alignment in accordance with a kabat numbering rule in a preferred embodiment.

The term “agglutination” as used herein refers to cell agglomeration, and the term “hemagglutination” refers to agglomeration of a particular class of cells (i.e., red blood cells) . Therefore, hemagglutination is a type of agglutination.

The control antibody “Hu5F9” herein is an anti-CD47 antibody in the form of IgG4P, formed by recombinant expression by GenScript according to the variable region sequence of 5F9 disclosed in patent US 2015/0183874 A1. The control antibody “SRF231” is an anti-CD47 antibody in the form of IgG4P, formed by recombinant expression by GenScript according to the variable region sequence of 2.3D11 disclosed in patent US 20180201677 A1.

Pharmaceutical formulation

The pharmaceutical formulation of the present invention is stable. A variety of methods well known in the art can be used to measure the stability of antibody formulations. For example, the purity of the antibody formulation can be analyzed and the aggregation level of the antibody can be evaluated by methods such as non-reduced CE-SDS and SEC-HPLC; charge variants in the antibody formulation can be analyzed by capillary isoelectric focusing electrophoresis (cIEF) , imaged capillary isoelectric focusing electrophoresis (iCIEF) , ion exchange chromatography (IEX) , etc. ; and the antibody/antigen binding can be detected by ELISA, FACS, or BIACORE, etc.

In one embodiment, after storage at a high temperature of 40℃ for at least 4 weeks, repeated freezing and thawing at -70℃/5℃, and/or storage at 2℃-8℃ for at least 6 months (preferably for at least one year, more preferably for at least two years) , the antibody formulation of the present invention remains clear, slightly opalescent, or free of visible particles.

In one embodiment, after storage at a high temperature of 40℃ for at least 4 weeks, repeated freezing and thawing at -70℃/5℃, and/or storage at 2℃-8℃ for at least 6 months (preferably for at least one year, more preferably for at least two years) , the purity of the anti-CD47 antibody in the antibody formulation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as determined by size exclusion chromatography or non-reduced CE-SDS.

In one embodiment, after storage at a high temperature of 40℃ for at least 4 weeks, repeated freezing and thawing at -70℃/5℃, and/or storage at 2℃-8℃ for at least 6 months (preferably for at least one year, more preferably for at least two years) , at least 50%, preferably at least 55%of the anti-CD47 antibody in the antibody formulation of the present invention is in a non-basic or a non-acidic form (i.e., main peak or main charge form) , as determined by imaged capillary isoelectric focusing electrophoresis.

In one embodiment, after storage at a high temperature of 40℃ for at least 4 weeks, repeated freezing and thawing at -70℃/5℃, and/or storage at 2℃-8℃ for at least 6 months (preferably for at least one year, more preferably for at least two years) , the change in biological activity percentage of the anti-CD47 antibody in the antibody formulation of the present invention does not exceed 30%, according to the specific antigen-binding activity of the antibody as determined by ELISA.

The present invention provides a stable pharmaceutical formulation, comprising (i) an anti-CD47 antibody or an antigen-binding fragment thereof; (ii) a buffer; (iii) a stabilizer; and (iv) a surfactant.

(I) Anti-CD47 antibody

The anti-CD47 antibody suitable for the pharmaceutical formulation of the present invention can be any anti-CD47 antibody. In some embodiments, "anti-CD47 antibody" described herein referred to an antibody, which can bind to CD47 protein with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD47. In some embodiments, the anti-CD47 antibody provided herein has a dissociation constant (KD) of ≤ 100 nM, ≤ 10 nM, ≤ 5 nM, ≤ 4 nM, ≤ 3 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.9 nM or ≤0.8 nM. In some embodiments, the antibody binds to a full-length human CD47 or fragment thereof (especially an extracellular binding fragment thereof) . In some embodiments, the antibody binds to a protein comprising a full-length CD47 or fragment thereof. In some other embodiments, the antibody binds to CD47 or a fragment thereof expressed on a cell surface.

In some embodiments, the anti-CD47 antibody provided herein can block the interaction between CD47 and SIRPα, has a high anti-tumor activity, and does not induce a significant red blood cell agglutination reaction.

In some embodiments, the anti-CD47 antibody is the anti-CD47 antibody described in Chinese Application 202010282924.0 (submitted on April 10, 2020) . For the purpose of this application, the contents of the Chinese application are incorporated herein by reference in its entirety.

The accurate amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined by using any one of many well known schemes such as Kabat, Chothia, AbM, Contact or North. It should be noted that the boundary of CDR of the variable region of the same antibody obtained by different definition systems may be different. That is, the CDR sequences of the variable region of the same antibody defined by different assignment systems are different. Therefore, when it comes to defining an antibody with a specific CDR sequence as defined in the present invention, the scope of the antibody also covers an antibody, the variable region sequence of which comprises the specific CDR sequence. However, due to the application of different schemes (such as different assignment systems or combinations) , the claimed CDR boundary is different from the specific CDR boundary as defined in the present invention.

In some embodiments, the CDR boundary of the anti-CD47 antibody molecule described here is determined based on the Kabat assignment system.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDR is different from antibody to antibody, only a limited number of amino acid positions in CDR are directly involved in antigen binding. The minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM and North methods to provide a “minimum binding unit” for antigen binding. The minimum binding unit can be a subset of CDR. As is appreciated by a person skilled in the art, the residues of the rest of the CDR sequence can be determined according to the structure and protein folding of the antibody. Therefore, the present invention also contemplates any variants of the CDR presented herein. In some embodiments, in a variant of CDR of the anti-CD47 antibody or antigen-binding fragment thereof described herein, the amino acid residue of the minimum binding unit can remain unchanged, while the residues of the rest of the CDR defined according to Kabat or IMGT can be replaced by conservative amino acid residues.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein comprises one to three selected from heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 11 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 12 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein comprises one to three selected from light chain complementary determining region 1 (HCDR1) , LCDR2 and LCDR3, wherein the LCDR1 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 14 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 22 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 22, and the LCDR3 comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 16 or has at least 1 and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein also covers an antibody or an antigen-binding fragment thereof, in the three CDRs of the heavy chain variable region of which, relative to the three CDRs specifically disclosed herein, a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) is comprised, and/or in the three CDRs of the light chain variable region of which, relative to the three CDRs specifically disclosed herein, a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) is comprised.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein also covers such an antibody or antigen-binding fragment thereof, wherein compared with the heavy chain variable region and/or light chain variable region of the antibody specifically disclosed herein, there are one or more (preferably no more than 10, more preferably no more than 6, 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) in the heavy chain variable region and/or light chain variable region, and preferably, the amino acid change does not occur in the CDR region.

In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence selected from SEQ ID NO: 1, 3, 5, 6 or 7. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof described herein comprises a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence selected from SEQ ID NO: 2, 4, 8, 9 or 10.

In an embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes of the present invention occur in regions outside CDRs (for example, in FRs) . More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region. In some embodiments, the amino acid changes occur in a heavy chain constant region and/or a light chain constant region.

In a preferred embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In a preferred aspect, conservative substitutions are derived from the conservative substitution residues shown in Table A below, preferably, the preferred conservative amino acid substitution residues shown in Table A.

Table A

In some embodiments, the antibodies of the present invention comprising amino acid changes have comparable or similar properties to the specific antibodies disclosed herein.

In some embodiments, the anti-CD47 antibody of the present invention includes post-translational modifications to CDRs, light chain variable regions, heavy chain variable regions, light chains, or heavy chains.

In some embodiments, the anti-CD47 antibody provided by the present invention is a full-length antibody, a single-domain antibody such as a VHH, a Fab antibody, a Fab’ antibody, a Fab’ -SH, a (Fab’ ) ₂ antibody, a single-chain antibody such as a scFv, a Fv, a dAb (domain antibody) or a bis (multi) -specific antibody.

In some embodiments, the anti-CD47 antibody provided by the present invention is any antibody in the form of IgG, such as an antibody in the form of IgG1, IgG2, IgG3 or IgG4. In some embodiments, the anti-CD47 antibody of the present invention is an antibody in the form of IgG4P, i.e., a modification, Ser228Pro (S228P, numbered according to EU) is carried out in the hinge region of the human IgG4 constant region to avoid or reduce chain exchange.

In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided by the present invention to produce Fc region variants. The Fc region variant can comprise a human Fc region sequence (such as the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications (such as substitutions) at one or more amino acid positions, for example, a number of modifications to human IgG1 to enhance or reduce its binding to FcγR and enhance or reduce the corresponding function are summarized in article of Bruhns and

published in Immunol Rev. 2015 Nov; 268 (1) : 25-51, page 44.

In some embodiments, the anti-CD47 antibody provided by the present invention comprises an Fc region variant, which has a Fc γ R binding activity which is reduced or deficient. In some embodiments, the Fc region variant has amino acid substitutions, and in particular, the amino acid substitutions are selected from other amino acid substitutions at positions E233, L234, L235, N297, and P331 of an immunoglobulin heavy chain. In some embodiments, the amino acid substitutions of the Fc region variant are E233P, L234A, L235A, L235E, N297A, N297D or P331S.

In some embodiments, the antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites of an antibody can be conveniently achieved by changing the amino acid sequence so as to produce or remove one or more glycosylation sites. Glycosylation can be changed, for example, to increase affinity of the antibody for the “antigen” . This modification can be accomplished, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which results in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at this site. This aglycosylation can increase affinity of the antibody for the antigen. Such a method is described in, for example, U.S. Patent No. 5,426,300. When the antibody comprises an Fc region, the saccharides attached to same can be changed. In some applications, modifications to remove undesired glycosylation sites are useful, such as removal of fucose modules to improve antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In other applications, galactosylation modification can be made to modify complement-dependent cytotoxicity (CDC) .

In some embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs, ” in which one or more residues of an antibody are substituted with cysteine residues.

In some embodiments, the antibody herein may be further modified to comprise additional non-protein moieties that are known in the art and readily available. The non-protein moieties include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , ethylene glycol/propylene glycol co-polymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dialkane, poly-1, 3, 6-trialkane, ethylene/maleic anhydride co-polymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol) , polyvinyl alcohol, and mixtures thereof.

Immunoconjugate and immune fusion

In some embodiments, the anti-CD47 antibody in the pharmaceutical formulation of the present invention can be replaced with an immunoconjugate, wherein the immunoconjugate comprises any anti-CD47 antibody or an antigen-binding fragment thereof provided herein and other substances. In one embodiment, the other substances are, for example, a cytotoxic agent, which includes any agent that is harmful to cells.

In some embodiments, the present invention provides a pharmaceutical formulation comprising an immune fusion comprising any anti-CD47 antibody or an antigen-binding fragment thereof provided herein.

(II) Buffer

As used herein, the term “buffer” denotes a pharmaceutically acceptable excipient, which is used to stabilize the pH of the pharmaceutical formulation. Suitable buffers are well-known in the art, and include but are not limited to a histidine buffer, a citrate buffer, a succinate buffer, an acetate buffer, an arginine buffer, a phosphate buffer or a mixture thereof. The buffer provided by the present invention is at a concentration of about 10 mM to about 80 mM, preferably about 10 mM to about 50 mM. In one embodiment, the buffer is at a concentration of about 10 mM, about 20 mM, about 30 mM, about 40 mM or about 50 mM, preferably about 20 mM.

The “citrate buffer” is a buffer comprising citrate ions. The citrate buffer may comprise one or more of citric acid, disodium citrate, potassium citrate and the like. In some embodiments, the citrate buffer is a citric acid -disodium citrate buffer.

The “histidine buffer” refers to a buffer comprising histidine ions. The histidine buffer may comprise one or more of histidine, histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate and the like. In some embodiments, the histidine buffer is a histidine-histidine hydrochloride buffer.

Independently from the buffers used, acids or bases (such as hydrochloric acid, acetic acid, phosphoric acid and citric acid, sodium hydroxide and potassium hydroxide) known in the art can be used to adjust the solution pH to about 5.0-7.0. In one embodiment, the pH is adjusted to about 5.0-7.0, about 5.0-6.5, about 5.0-6.0, about 5.5- 7.0, about 6.0-7.0, or about 6.5-7.0; preferably, the pH is adjusted to about 5.0, about 5.5, about 6.0, about 6.5, about 7.0; more preferably, the pH is adjusted to about 6.5.

In some embodiments, the buffer in the pharmaceutical formulation of the present invention is an acetate buffer with a concentration of about 20 mM, and the pH is about 5.0-6.5, preferably about 5.0 or about 5.5. In one embodiment, the buffer is a citrate buffer (e.g., citric acid -disodium citrate buffer) with a concentration of about 20 mM, and the pH is about 5.0-6.5, preferably about 6.0 or about 6.5. In one embodiment, the buffer in the pharmaceutical formulation of the present invention is a phosphate buffer with a concentration of about 20 mM, and the pH is about 5.0-6.5, preferably about 6.5. In one embodiment, the buffer in the pharmaceutical formulation of the present invention is a histidine salt buffer (e.g., histidine-histidine hydrochloride buffer) with a concentration of about 20 mM, and the pH is about 5.0-6.5, preferably about 5.5, about 6.0, or about 6.5, more preferably about 6.5.

(III) Stabilizer

As used herein, the term “stabilizer” denotes a pharmaceutically acceptable excipient, which is used to protect the active pharmaceutical ingredient and/or the formulation from chemical and/or physical degradation during production, storage and application.

The pharmaceutical formulation of the present invention comprises at least one stabilizer, which can be selected from an amino acid, a sugar, a polyol, a salt, and a combination thereof.

As used herein, the term “amino acid” denotes a pharmaceutically acceptable organic molecule possessing an amino moiety at the α-position of a carboxylic group. Examples of the amino acids include but are not limited to arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine, serine, and proline. The amino acid employed in various cases is preferably an L-amino acid. Basic amino acids, such as arginine, histidine, or lysine, are preferably in the form of inorganic salts thereof (advantageously in the form of hydrochlorides, i.e., as amino acid hydrochlorides) . A preferred amino acid used in the present invention is arginine, preferably arginine hydrochloride. The concentration of arginine hydrochloride used is about 1 mg/mL to about 100 mg/mL, about 5 mg/mL to about 40 mg/mL, such as about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 26 mg/mL, about 32 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, or about 90 mg/mL, more preferably about 26 mg/mL.

As used herein, the term “sugar” includes a monosaccharide and an oligosaccharide. A monosaccharide is a monomeric carbohydrate which cannot be hydrolyzed by acids, including simple sugars and derivatives thereof, e.g., aminosugars. Sugars in D conformation are usually used. Examples of the monosaccharide include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. An oligosaccharide is a carbohydrate consisting of more than one monomeric saccharide unit connected via branched or linear glycosidic bond (s) . The monomeric saccharide units within an oligosaccharide can be identical or different. Depending on the number of monomeric saccharide units, the oligosaccharide is a di-, tri-, tetra-, or penta-saccharide, etc. In contrast to a polysaccharide, the monosaccharide and the oligosaccharide are water soluble. Examples of the oligosaccharide include sucrose, trehalose, lactose, maltose and raffinose. A preferred sugar used in the present invention is sucrose and trehalose (i.e., α, α-D-trehalose) , most preferably, sucrose. Trehalose is available as trehalose dihydrate. In some embodiments, the concentration of the sugar (such as sucrose) in the pharmaceutical formulation is about 10 mg/mL to about 100 mg/mL, preferably about 20 mg/mL to about 80 mg/mL, such as about 20 mg/mL, about 30 mg/mL, about 50 mg/mL, about 65 mg/mL, or about 80 mg/mL.

As used herein, the term “polyol” denotes pharmaceutically acceptable alcohols having more than one hydroxy group (s) . Suitable polyols include but are not limited to mannitol, sorbitol, glycerol (glycerine) , dextran, arabitol, propylene glycol, polyethylene glycol, and combinations thereof. In some embodiments, the concentration of the polyols (such as mannitol, sorbitol) in the pharmaceutical formulation is about 20 mg/mL to about 100 mg/mL. In some embodiments, the concentration of the polyols (such as mannitol, sorbitol) in the pharmaceutical formulation is about 40 mg/mL to about 60 mg/mL, preferably about 45 mg/mL.

The “salt” in the pharmaceutical formulation of the present invention refers to an inorganic salt, such as sodium chloride, magnesium chloride, and calcium chloride.

In some embodiments, the stabilizer in the pharmaceutical formulation of the present invention is an amino acid, preferably arginine. In one embodiment, the stabilizer is a combination of an amino acid and a sugar; and preferably, the amino acid is arginine, and the sugar is sucrose or trehalose. In one embodiment, the stabilizer is a combination of an amino acid and a polyol; and preferably, the amino acid is arginine, and the polyol is sorbitol or mannitol. In one embodiment, the stabilizer is a combination of an amino acid and a salt; and preferably, the amino acid is arginine, and the salt is sodium chloride.

In some embodiments, the stabilizer in the pharmaceutical formulation of the present invention is arginine, wherein the concentration of arginine is about 5 mg/mL to about 40 mg/mL, preferably about 26 mg/mL and about 32 mg/mL. In one embodiment, the stabilizer in the pharmaceutical formulation of the present invention is a combination of arginine and sucrose, wherein the ratio of mass concentration of arginine to sucrose is about 1 : 1 to about 1 : 20, such as about 1 : 1, about 1 : 2, about 1 : 3, about 1 : 4, about 1 : 5, about 1 : 6, about 1 : 7, about 1 : 8, about 1 : 9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 : 13, about 1 : 14, about 1 : 15, about 1 : 16, about 1 : 17, about 1 : 18, about 1 : 19, and about 1 : 20.

In one embodiment, the pharmaceutical formulation of the present invention is isotonic.

(IV) Surfactant

The pharmaceutical formulation of the present invention can also comprise a surfactant. The term “surfactant” generally includes a reagent that protects the protein, e.g., an antibody, from air/solution interface-induced stresses, and solution/surface induced-stresses, to reduce aggregation of the antibody, or to minimize the formation of particulates in the formulation. The surfactant in the pharmaceutical formulation of the present invention can be nonionic surfactants such as polysorbates (e.g., polysorbate-20 and polysorbate-80) or poloxamers (e.g., poloxamer-188) .

The amount of the surfactant in the pharmaceutical formulation of the present invention can vary with the specific characteristics of interest of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some embodiments, the concentration of the surfactant (such as polysorbate-80) in the pharmaceutical formulation is about 0.002% (w/v) (i.e., 0.02 mg/mL) to about 0.5% (w/v) (i.e., 5 mg/mL) , about 0.01% (w/v) (i.e., 0.1 mg/mL) to about 0.1% (w/v) (i.e., 1 mg/mL) . In some embodiments, the concentration of the surfactant (such as polysorbate-80) in the pharmaceutical formulation is about 0.01% (w/v) (i.e., 0.1 mg/mL) to about 0.05% (w/v) (i.e., 0.5 mg/mL) , preferably about 0.01% (w/v) (i.e., 0.1 mg/mL) , about 0.02% (w/v) (i.e., 0.2 mg/mL) , or 0.03% (w/v) (i.e., 0.3 mg/mL) , more preferably about 0.02% (w/v) (i.e., 0.2 mg/mL) .

For other considerations, other excipients can also be used in the formulation of the present invention. The excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, and alum. These and other known pharmaceutical excipients and/or additives suitable for use in the formulation of the present invention are well known in the art, for example, as listed in “The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe et al, American Pharmaceuticals Association (2003) ; and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams and Wilkins (2005) ” .

The pharmaceutical formulation according to the present invention can also be provided in a lyophilized form or in a liquid form reconstituted from the lyophilized form. The “lyophilized form” is prepared by freeze-drying methods known in the art. The lyophilizate usually has a residual moisture content of about 0.1% (w/v) to about 5% (w/v) and is present in a powder or a physically stable cake form. The “reconstituted form” can be obtained from the by fastly dissolving lyophilizates after the addition of a reconstitution medium. Suitable reconstitution media include but are not limited to water for injection (WFI) , bacteriostatic water for injection (BWFI) , a sodium chloride solution (such as 0.9% (w/v) NaCl) , and a glucose solution (such as 5% (w/v) glucose) .

Preparation of formulation

Any of the anti-CD47 antibodies described herein can be produced using methods known in the art, for example, by a method comprising culturing a host cell (HEK293 cells, or HEK293T, HEK293F, and HEK293E cells obtained by means of modification on the basis of HEK293 cells; CHO cells or CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO cells obtained by means of modification on the basis of CHO cells) containing the nucleic acid encoding any of the anti-CD47 antibodies described herein in a form suitable for expression, under conditions suitable to produce this type of antibodies, recovering the cell broth, and purifying the antibody using conventional purification methods.

Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering, 99 (2008) 599-613) describes an antibody three-column purification method in which ion exchange chromatography (anionic IEX and/or cationic CEX chromatography) is used after a protein A capture step. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. Biotechnol Bioeng. 2008 Oct 15; 101 (3) : 553-66. ) describes a two-column purification method in which a weak partitioning anion exchange resin is used after a protein A affinity chromatography.

After the antibody with a sufficiently high purity is obtained, a formulation comprising the antibody can be prepared according to methods known in the art. For example, the preparation can be performed using the following steps: (1) centrifuging and clarifying the fermentation broth after the fermentation to remove impurities such as cells to obtain a supernatant; (2) capturing the antibody using affinity chromatography (for example, a protein A column with specific affinity for IgG1, IgG2 and IgG4 antibodies) ; (3) inactivating the virus; (4) refining and purifying same (CEX cation exchange chromatography can be used generally) to remove impurities in the protein; (5) filtering the virus (to reduce the virus titer by, e.g., 4log10 or more) ; and (6) ultrafiltering/diafiltering same (which can be used to displace a protein to a formulation buffer which is favorable for the stability of the protein and concentrate same to a suitable concentration for injection) . Minow B, Rogge P, Thompson K. Implementing a Fully Disposable MAb Manufacturing Facility. BioProcess Int. 10 (6) 2012: 48–58.

Treatment methods or uses

In one aspect, the present invention provides a method of preventing, diagnosing or treating CD47-related diseases in a subject. The method comprises administering an effective amount of the pharmaceutical formulation described herein to a patient in need thereof.

In one aspect, the present invention provides the use of the anti-CD47 antibodies or the antigen-binding fragments thereof in the production or preparation of a pharmaceutical formulation for the prevention, diagnosis or treatment of CD47-related diseases in a subject.

In one aspect, the pharmaceutical formulation provided by the present invention can be used to prevent or treat CD47-related diseases in a subject. For CD47-related diseases in a subject identified by using standard methods, the pharmaceutical formulation disclosed by the present invention can be administered.

In some embodiments, the CD47-related diseases of the present invention refer to diseases related to abnormal CD47 expression, activity and/or signal transmission in a subject, including but not limited to cancers. In some embodiments, in CD47-related diseases, the (level or content) of nucleic acid encoding CD47 is increased, or CD47 expression is increased, or CD47 protein level is increased, or activity is increased, or activity signal transmission is increased.

In some embodiments, the treatment of the diseases will benefit from the inhibition of CD47 in nucleic acid or protein levels, or benefit from blocking of the binding of CD47 to its ligand or CD47-mediated signal transmission.

The term “subject” or “patient” or “individual” herein includes any human or non-human animals. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, bovine, chicken, amphibians, reptiles, etc. In some embodiments, the subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or having a risk of suffering from a disease described herein) . In one embodiment, the subject suffers from or has a risk of suffering from a disease described herein (e.g., a cancer) . In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy.

In some embodiments, the cancer includes various hematological cancer and solid tumors, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be at an early stage, a middle stage or a late stage, or a metastatic cancer. The cancer is, for example, bladder cancer, pancreatic cancer, lymphomas, leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcomas, glioma, glioblastoma, myeloma, endometrial cancer, renal carcinoma, (benign) melanoma, prostate cancer, thyroid carcinoma, cervical cancer, gastric cancer, or liver cancer. In some embodiments, the lymphoma is selected from Burkitt lymphoma, diffuse large cell lymphoma, or mantle cell lymphoma. In some embodiments, the leukemia is promyelocytic leukemia.

The pharmaceutical formulation of the present invention may be administered in any suitable manner, including oral, parenteral, intrapulmonary and intranasal administration, and, if topical treatment is needed, it can be administered intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various administration regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

In some embodiments, the pharmaceutical formulation of the present invention can be administered in combination with another therapeutic agent to a subject or an individual for the treatment of a disease. For example, for the treatment of cancers, the pharmaceutical formulation herein can be administered in combination with another anti-cancer treatment (such as chemotherapy or treatment with a different antibody) . In some embodiments, the therapeutic agents are, for example, chemotherapeutic agents, radio therapeutic agents, cytokines, vaccines, other antibodies, immunomodulators or other biomacromolecular drugs. In some embodiments, the therapeutic mode includes surgery; and radiation therapy, local irradiation or focus irradiation, etc. The above-mentioned combination therapy includes combined administration (in which two or more of therapeutic agents are contained in the same or separate preparations/formulations) and separate administration, wherein the administration of the pharmaceutical formulation of the present invention may occur prior to, simultaneously with, or after administration of additional therapeutic agent and/or adjuvant and/or treatment.

The “therapeutically effective amount” of the antibody will depend, for example, on the condition to be treated, the severity and course of the condition, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, the type of the antibody used, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The antibody may be administered as the sole treatment or in combination with other drugs or therapies which is useful in treating the condition in question.

As a general proposition, the therapeutically effective amount of the antibody administered to human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations. In some embodiments, the antibody used is administered daily, for example, at about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at about 15 mg/kg. However, other dosage regimens may also be useful. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses) , such as via infusion. The dose of the antibody administered in a combination therapy may be reduced compared with a single-agent therapy. The progress of this therapy is easily monitored by conventional techniques.

Article of manufacture or kit

The present invention provides an article of manufacture or a kit, comprising a container filled with the pharmaceutical formulation of the present invention, and optionally provides instructions for using same. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be prepared from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin) , or metal alloy (such as stainless steel or Hastelloy C gauze) . The container is filled with the pharmaceutical formulation and the label on, or associated with, the container may indicate instructions for use. The article of manufacture may further include other materials which are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., a chemotherapeutic agent, and an anti-neoplastic agent) . Suitable containers for the one or more agents include, for example, bottles, vials, bags and syringes.

Combination product

In one aspect, the present invention also provides a combination product, comprising the pharmaceutical formulation of the present invention, and one or more additional therapeutic agents. The combination product of the present invention can be used in the treatment method of the present invention.

The present invention includes any combinations of specific embodiments described herein. It should be understood that although the specific content and examples are described to illustrate the preferred embodiments of the present invention, these are merely illustrative and used as examples. The present invention further covers embodiments modified on the basis of the preferred embodiments of the present invention that are obvious to a person skilled in the art. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety.

Detailed Description of Embodiments

Preparation and activity verification of the antibodies in the pharmaceutical formulation of the present invention

Example 1 Preparation and screening of hybridoma-derived antibody

The anti-CD47 antibodies were obtained by hybridoma technique. The recombinant protein CD47-Fc (ACROBiosystems, Cat: CD7-H5256) containing the extracellular domain of human CD47 with a Fc tag was used as an antigen to immunize mice. After mixing and emulsifying the recombinant protein CD47-Fc with complete or incomplete Freund’s adjuvant (Sigma-Aldrich) , SJL mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) and BALB/c mice (Yangzhou University Medical Center) were immunized. The mice were subjected to one round of immunization (complete Freund’s adjuvant) and two rounds of booster immunization (incomplete Freund’s adjuvant) and taken blood after each booster immunization. The binding activityof the serum of the mice after immunization to the recombinant human CD47-Fc (ACROBiosystems, Cat: CD7-H5256) protein is detected by ELISA assay, and at the same time, the binding potency of mice serum to CHO cells (constructed by GenScript) overexpressing human CD47 was detected by flow cytometry (FACS) . Spleen cells of the mice with a higher serum titer were selected to fuse with myeloma cell line SP2/0 (ATCC) . Four days before fusion, the recombinant protein CD47-Fc of human CD47 extracellular domain was intraperitoneally injected into mice for booster immunization. On the day of fusion, mice were euthanized, and then mouse spleen cells were homogenized to obtain a single cell suspension. The mouse spleen cells were fused with murine myeloma cell line SP2/0 (3: 1) by means of an electrofusion apparatus. The fused cells were resuspended in a medium containing HAT (hypoxanthine, aminopterin and thymidine deoxynucleotide, GIBCO, Cat: 21060016) to screen the successfully fused hybridoma cells. The supernatant of hybridoma cells was collected and the hybridoma cells that secreted antibodies specifically binding to human CD47 were screened by two rounds of ELISA. Then, the activity of secretion supernatant of the hybridoma was determined by CD47-related functional screening tests (such as binding specificity with human CD47 or cynomolgus monkey CD47; no activity in induction of red blood cell agglutination; activity in promoting phagocytosis of tumor cells by macrophages) , and then the positive hybridoma clones were selected and subcloned for single or multiple rounds to obtain monoclone. After screening, 125G4A4 was finally chosen as a hybridoma clone.

The candidate hybridoma cell 125G4A4 was subjected to an expanded culture, and after 7-10 days of culturing, the supernatant was collected, centrifuged and filtered to remove cells and debris. The supernatants were passed through a Protein A purification column (GenScript) , then cleaned and equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0) , and then eluted with 0.1 M sodium citrate (pH 3.5) ; and the eluent was immediately neutralized with one ninth volume of 1 M Tris-HCl (pH 9) , and then dialyzed with PBS buffer. Finally, the hybridoma-derived antibody 125G4A4 was obtained for further characterization.

1.1 Detection of binding activities of antibodies to CHO-K1 cells overexpressing human CD47 proteins by FACS

A human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in hamster ovary cell line CHO-K1 to establish CHO-K1 cell line overexpressing the human CD47 protein. The cells were co-incubated with serially diluted antibody 125G4A4 and referenceantibody C0774CK230-C (i.e., Hu5F9) (the highest concentration being 300 nM, three fold dilution, 12 concentration points in total) at 4℃ for 50 minutes. After washing twice with iced PBS, the cells were incubated with an iFluor647-labled goat anti-mouse IgG (H + L) antibody (Genscript) at 4℃ in the dark for 40 minutes. The cells were washed twice with iced PBS, and then the fluorescence signal was detected by Calibur (BD Biosciences) flow cytometry, and according to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated. As shown in Table 1, the finally obtained hybridoma-derived antibody 125G4A4 has a high binding activity to CHO-K1 overexpressing human CD47 protein, with an EC ₅₀ of 0.22 nM.

1.2 Detection of binding activities of antibodies to CD47 on the surface of tumor cells by FACS

Human CD47 was endogenously expressed on the cell surface of human Burkitt lymphoma cell line Raji. The antibody 125G4A4 and the reference antibody Hu5F9 were serially diluted into PBS containing 2%fetal bovine serum (FBS, Gibco, Cat: 10100147) (the highest concentration being 46.3 nM, three fold dilution, 8 concentration points in total) . The diluted antibodies were mixed with and co-incubated with Raji cells (purchased from ATCC) (5*10 ⁵ cells /well) at 4℃ for 1 hour. After washing three times with PBS containing 2%fetal bovine serum (FBS) , a PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added and incubated with the cells at 4℃ in the dark for 1 hour. The cells were washed three times with PBS containing 2%fetal bovine serum (FBS) , and then the fluorescence signal was detected by CantoII (BD Biosciences) flow cytometry, and according to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated. As shown in Table 1, the hybridoma-derived antibody 125G4A4 has a binding activity to Raji cells, with an EC ₅₀ of 0.84 ± 0.02 nM.

1.3 Blockade of the interaction between human CD47 and SIRPα by anti-CD47 antibodies

ELISA assay was performed to detect the ability of 125G4A4 to block the interaction between human CD47 and SIRPα. The recombinant protein hCD47-Fc containing the extracellular domain of human CD47 fused with the Fc fragment of human IgG (ACROBiosystems, Cat: CD7-H5256) was coated onto a 96-well plate and incubated overnight at 4℃. After the plate was washed 3 times with PBST (PBS containing 0.5%Tween-20) , PBST containing 1%BSA was added for blocking the plate for 2 hours. After the plate was washed three times with PBST, the mixture of serially diluted antibody 125G4A4 or reference antibody Hu5F9 (the highest concentration being 66.7 nM, three fold dilution, 8 concentration points in total) together with SIRPα-His recombinant protein (ACROBiosystems, Cat: SIA-5225) with a final concentration of 2.5 μg/ml was added and incubated at room temperature for 1 hour. The plate was washed three times with PBST, and a horseradish peroxidase labeled goat anti-His-tag secondary antibody (CWBIO, Cat: CW0285M) was added to detect SIRPα captured by coated CD47 protein. After the 96-well plate was incubated at 37℃ for 30 minutes, the plate was washed 5 times with PBST, and a TMD (Surmodics, Cat: TMBW-1000-01) developing solution was added, and was incubated in the dark for 15 minutes. 2N H ₂SO ₄ was added to terminate the color-developing reaction. OD ₄₅₀ was read on a microplate reader. The absorbance value reflected the amount of SIRPα which bound to CD47. Graphpad was used for fitting a concentration dependent curve, and the IC ₅₀ of anti-CD47 antibody for blocking binding of CD47 to SIRPα was calculated. As shown in Table 1, 125G4A4 can effectively block the CD47/SIRPα interaction, with an IC ₅₀ of 3.06 nM.

1.4 Detection of activity of anti-CD47 antibodies in induction of human red blood cell agglutination

It is known that in the prior art, most of the anti-CD47 antibodies have the property of inducing red blood cell agglutination. It is widely believed that the property is closely related to clinical side effects such as anemia existing in the treatment by therapeutic anti-CD47 antibody. Therefore, we evaluate the anti-CD47 antibody in the present invention by a red blood cell agglutination experiment in vitro to screen the antibody without the property of inducing red blood cell agglutination. The method is as follows: collecting the healthy donor’s fresh human blood, washing the cells five times with PBS, and then diluting the cells to make a suspension containing 10%human red blood cells; mixing the red blood cell suspension with the experimental antibody (antibody 125G4A4 and reference antibody Hu5F9, the highest concentration being 667 nM, three fold dilution, 12 concentration points in total) , then adding the mixture into a round bottom 96-well plate; and incubating them at room temperature for 16 hours, then taking photos and determining the results according to the phenomenon of the cells in the well. If red blood cell agglutination occurs, cells are plated onto each well like a net, and a larger sheet-like cell layer will appear in the well with a diameter larger than that of the negative control well; on the contrary, if no hemagglutination occurs, the red blood cells will deposit at the bottom of the well, and smaller dot-like cell pellete precipitation will appear in the well. 125G4A4 shows no obvious phenomenon of inducing red blood cell agglutination in the experiment.

1.5 Determination of the pro-phagocytic effects of anti-CD47 antibodies on tumor cells by human macrophages

The ability of antibody 125G4A4 of the present invention to promote phagocytosis of tumor cells by macrophages was detected by assay based on flow cytometry. Human blood was freshly collected from healthy donors, and the peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) . Monocytes were further isolated and obtained by using the human total monocyte Isolation Kit (Miltenyi biotec, cat: 130-096-537) . To induce the monocytes to differentiate into macrophages, macrophage colony stimulating factor (M-CSF, R &D Systems, Cat: 216-MC) was added and the monocytes was subjected to adherent culture for 7 consecutive days. On the day of cellular phagocytosis experiment, the above-mentioned differentiated macrophages were starved in a serum-free medium for 2 hours. At the same time, target tumor cells Raji were flurorescent labeled with CFSE (eBioscience, Cat: 65-0850-85) according to the steps recommended by the instructions. The CFSE-labeled tumor cells and macrophages were mixed in a ratio of 4 : 1, and the experimental antibodies of a detected concentration were added and incubated at 37℃ for 2 hours. Then the cells were washed twice with PBS, and then digested with trypsin (Gibco, Cat: 25200072) ; an APC labeled anti-CD14 antibody (Biolegend, Cat: 325608) was added and incubated in the dark on ice in PBS containing 2%fetal bovine serum for 30 minutes. The cells were washed twice and analyzed by flow cytometry. The percentage of CFSE positive cells in CD14 positive macrophage populations was calculated. As shown in Table 1, 125G4A4 can effectively promote the phagocytic function of macrophages on tumor cells.

Table 1 Determination of the Activity and function of hybridoma-derived antibody 125G4A4

Example 2 Humanization of hybridoma-derived antibody

2.1 Determination of variable region sequence of hybridoma-derived antibody

According to the method for hybridoma sequencing, the cells of hybridoma clone 125G4A4 were subjected to an expanded culture; total RNA was extracted with TRIzol (purchased from Ambio) and reverse transcribed into DNA with antibody-specific primers (Takara, PrimerScript 1 ^st Strand cDNA Synthesis Kit) ; and a gene fragment encoding mouse immunoglobulin V-region was subjected to amplification with antibody-specific primers. The variable region sequence of hybridoma-derived antibody was obtained by sequencing analysis. The amino acid sequences of the heavy chain variable region and the light chain variable region of the 125G4A4 antibody are as set forth in SEQ. ID Nos: 1 and 2, respectively, and the nucleotide sequences are as set forth in SEQ. ID Nos: 19 and 20, respectively.

2.2 Construction and expression of chimeric antibodies

According to the mechanism of action of CD47, in a specific embodiment of the present invention, the constant region of human IgG4 (S228P) is used as the heavy chain constant region of the antibody, and the human κ light chain constant region chain is used as the light chain constant region of the antibody. Mutation of serine at position 228 of IgG4 core hinge region to proline (S228P) can enhance the disulfide bond connection in the core hinge region and reduce the exchange of IgG4 Fab arm, and thereby greatly reduce the formation of half molecules. After the genes encoding heavy chain and light chain constant regions were synthesized, the heavy chain and light chain variable region genes were homologously recombined into a vector PTT5 with double enzyme digestion by EcoRI and BamHI. After sequenced to be correct, the heavy chain and light chain of an antibody at a molar ratio of 1.5 : 1are co-transfected into HEK293 cells. After 120 hours of culture, the supernatant was collected by centrifugation and purified to obtain a chimeric antibody.

Before humanization design, it is necessary to mutate some post translational modification (PTM) sites in the CDR region to avoid affecting the protein conformation, thereby affecting the function thereof. According to PTM analysis, two PTM sites in the CDR of 125G4A4 were identified, including one NSS glycosylation site in the heavy chain and one DG isomerization site in the light chain. The NSS glycosylation site and DG isomerization sites were mutated into QSS and EG, respectively. The mutated chimeric antibody obtained by purification in this example is named as Ch-125G4-m35. The amino acid sequences of the heavy chain variable region and the light chain variable region of Ch-125G4-m35 antibody are as set forth in SEQ. ID Nos: 3 and 4, respectively.

2.3 Humanized design of chimeric antibodies

To select the human antibody backbone sequence with highest similarity to chimeric antibody 125G4A4m for humanization, the variable region sequence of chimeric antibody 125G4A4m was Blast aligned with the PDB Antibody database., The heavy chain variable region of 125G4A4 m has a higher sequence homology with human germline IGHV1-69, and the light chain variable region thereof has a higher sequence homology with human germline IGKV1-16. Then the amino acid sequence of the variable region CDR and the accurate boundary thereof are defined by the Kabat assignment system. Then, the CDR segments of the variable region of the murine antibody are grafted into the human backbone sequence to obtain the humanized antibody.

In order to maintain the activity of the humanized antibody, the framework amino acid sequences of the variable region and its surrounding region are analyzed with macromolecular docking analysis by using computer simulation technology to investigate their spatial stereoscopic binding mode. By calculating electrostatic force, van der Waals force, hydrophilicity and entropy, the key amino acid individuals that may interact with CD47 and maintain the spatial framework in the candidate antibody gene sequence are analyzed and grafted back to the selected human antibody gene framework. Meanwhile the amino acid positions in the framework region that must be reserved are marked. Based on the above process, the humanized antibody is synthesized. Some key sites in the antibody framework region were back mutated into the antibody framework region sequence of chimeric antibody Ch-125G4-m35. According to the number and arrangement of back mutations, a number of different humanized heavy chain variable regions (SEQ. ID No: 5, SEQ. ID No: 6, SEQ. ID No: 7) and light chain variable regions (SEQ. ID No: 8, SEQ. ID No: 9, SEQ. ID No: 10) were designed respectively (see Table 2) . The finally determined humanized antibody Hu-125G4A4-48 of the present invention was named as HMA02h14-48 hereafter. The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are as set forth in SEQ. ID Nos: 7 and 8, respectively.

Table 2 Number and arrangement of reverse mutations in 125G4A4m heavy chain and light chain

Table 3 Amino acid sequence of anti-CD47 antibody

Table 4 CDR amino acid sequences of anti-CD47 antibodies (Kabat definition)

2.4 Expression of humanized antibodies

The DNA fragments encoding the above-mentioned designed humanized heavy chain and light chain variable regions were amplified and cloned into a vector comprising a constant region expressing a human antibody to construct an antibody-expressing plasmid (pCDNA3.4, purchased from Thermo Cat#A14697) . The heavy and light chain expression vectors were co-transfected into Expire293 cells (Thermo Cat#A14525) . After culturing at 37℃ for 6 days, the supernatant was collected. According to the above-mentioned method, the recombinant antibody was obtained by protein A affinity purification for further characterization of the antibody. The humanized antibody is IgG4 S228P (IgG4P) subtype.

Example 3 Screening of humanized antibodies

Highly active humanized antibodies were screened by detecting the binding ability of humanized antibodies to cynomolgus monkey B cells, the ability of human macrophages to phagocytose tumor cells and the ability of induction of red blood cell agglutination.

Detection of the binding ability of the humanized antibody to cynomolgus monkey B cells: flow cytometry method was used to detect the binding of a series of humanized 125G4A4 antibodies to CD47 on the surface of cynomolgus monkey B cells. The method is as follows: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of cynomolgus monkeys (provided by Shanghai Yinuosi Bio-Technology Co., Ltd. ) by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02) . PBMC was incubated with a series of humanized 125G4A4 antibodies or isotypes control (IgG4P) in PBS containing 2%fetal bovine serum at 4℃ for 30 minutes. Then the cells were washed three times and incubated with the secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2%fetal bovine serum at 4℃ in the dark for 30 minutes. The cells were washed three times and analyzed by flow cytometry. B cells were labeled with an anti-human CD20 antibody (Brilliant Violet 421 ^TM labeled anti-human CD20 Antibody, Biolegend, Cat: 302330) having cross-reactivity with cynomolgus monkeys, and detected by flow cytometry on Canto II (BD Biosciences) to obtain its average fluorescence intensity (MFI) .

According to the methods described in Examples 1.5 and 1.4, the ability of macrophages to phagocytose tumor cells and the ability of induction of red blood cell agglutination were detected respectively.

As shown in Table 5, a series of humanized 125G4A4 antibodies bind to CD47 expressed on cynomolgus monkey B cells under tested concentration. The antibodies promote phagocytosis of tumor cells Raji by macrophages, of which Hu-125G4A4m-48 displays the strongest phagocytic efficiency at 33 nM. The other activities of Hu-125G4A4m-48 are similar to those of chimeric antibody Ch-125G4m-m35. Moreover, the number of back mutations was smaller. Therefore, Hu-125G4A4m-48 was selected for further test, and was named as HMA02h14-48 hereafter.

Table 5 In vitro activity test of a series of anti-CD47 humanized antibodies

Example 4 Determination of binding activity of HMA02h14-48 to tumor cells by FACS

Human CD47 is endogenously expressed on the surface of human Burkitt lymphoma cell line Raji cells (Shanghai Institutes for Biological Sciences, SIBS, CCL-86 ^TM/ATCC) , human diffuse large cell lymphoma Toledo cells (

CRL-2631 ^TM) and human mantle cell lymphoma REC-1 cells (

CRL-3004 ^TM) . According to the detection method described in the preceding Example 1.2, flow cytometry was used to detect the binding of the humanized antibody HMA02h14-48 to CD47 on the surface of the above-mentioned tumor cell lines. The highest antibody concentration was 667 nM, the antibodies were serially diluted, and a total of 8 concentration points were tested.

As shown in figures 1-3. Both HMA02h14-48 and Hu5F9 bound to CD47 on the surface of tumor cells, including Raji, Toledo and REC-1. The maximum fluorescence intensity when HMA02h14-48 reaches a plateau is higher than that of Hu5F9, and the EC ₅₀ and the maximum fluorescence intensity are shown in Table 6.

Table 6 Binding activity of antibody HMA02h14-48 to CD47 on the surface of tumor cells

The negative isotype control antibody (isotype) used in this example and other examples was human IgG4P, which was purchased from Shanghai Chempartner Co., Ltd.

Example 5: Determination of binding affinity of antibody HMA02h14-48 to human CD47 by Biacore

Biacore was used to determine the binding kinetic parameters by measuring surface plasmon resonance (SPR) . This technology was used to detect the microscopic rate constants of the binding (ka) and dissociation (kd) of an antibody and an antigen. Based on the ka and kd values, the affinity value of the antibody and the antigen can be obtained. Both Biacore instrument (Biacore T200) and reagents were purchased from GE Healthcare. The anti-human Fc antibody was immobilized on sensor chip CM5. The purified antibodies (HMA02H14-48 and Hu5F9) were diluted in a mobile phase buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Tween-20, pH 7.4) , and flowed through a CM5 chip coated with anti-human Fc antibodies. Then the serially diluted human CD47-His (ACROBiosystems, Cat: CD7-H5227) fusion protein flowed through a detection chip to measure the binding of the antigen and the antibody, and then the mobile phase buffer flowed through the chip to detect the dissociation of the antigen from the antibody. The binding and dissociation signal data of the antigen and the antibody were collected at different concentrations, and fitted at 1 : 1 by a Langmuir model to calculate the affinity of the antigen and the antibody.

As shown in Table 7, HMA02h14-48 binds to human CD47 with high affinity with a K _D value of 7.77E-10 (M) .

Table 7 Determination of kinetic constants of humanized antibody binding to human CD47 by Biacore

Example 6 Detection of activity of HMA02h14-48 in blocking the interaction between human CD47 and SIRPα by ELISA

According to the method described in the preceding example 1.3, ELISA was used to detect the ability of HMA02h14-48 to block the interaction between human CD47 and SIRPα. The highest antibody concentration was 67 nM, the antibodies were serially diluted, and a total of 8 concentration points were tested.

As shown in figure 4, the antibody HMA02h14-48 in the present invention blocks the interaction between human CD47 and SIRPα with IC ₅₀ = 1.58 nM.

Example 7 Effects of HMA02h14-48 on phagocytosis of tumor cells by human macrophages

According to the method described in Example 1.5, the effect of HMA02h14-48 on promoting phagocytosis of human Burkitt lymphoma cell line Raji cells, human diffuse large cell lymphoma Toledo cells, human mantle cell lymphoma REC-1 cells and human promyelocytic leukemia cell line HL-60 cells by human macrophages was detected. The highest antibody concentration was 100 μg/mL, the antibodies were serially diluted, and a total of 8 concentration points were tested. The results were shown in table 8 and figure 4-8.

The results showed that compared with the reference antibodies Hu5F9 and SRF231, HMA02h14-48 could effectively promote the phagocytosis of human Burkitt lymphoma cell line Raji by macrophages. The highest phagocytic efficiency was up to 36.5%, and the phagocytosis rates at different concentrations from 0.1 to 100 μg/ml were higher than those of Hu5F9 and SRF231. The highest phagocytic efficiency of HMA02h14-48 for human mantle cell lymphoma REC-1 was up to 84.6%, and the phagocytosis rate could be maintained at about 70%even at a low concentration of 0.1 μg/ml, which was higher than those of Hu5F9 and SRF231. HMA02h14-48 promoted the phagocytosis of Toledo cells by macrophages, and the phagocytosis rate was up to 94.2%. HMA02h14-48 could promote the phagocytosis of tumor cell HL-60 by macrophages, and the highest phagocytic efficiency was up to 65%.

Table 8 Effect of antibody HMA02h14-48 for promoting phagocytosis of tumor cells by human MΦ

Example 8 Detection of effects of HMA02h14-48 on the induction of red blood cell agglutination in vitro

Human red blood cells were diluted to 10%in PBS, and incubated with CD47 antibody added in a round bottom 96-well plate for 16 hours at room temperature. The presence of non-precipitated red blood cells is an evidence that proves red blood cell agglutination. Compared with the white dots formed by the precipitation of non-agglutinated red blood cells, the non-precipitated red blood cells could form a reticulated area which was larger than that of the negative isotype control antibody (see figure 9) . The results of the negative isotype control antibody (Isotype) were used as normal standards.

According to the method described in Example 1.4, the antibody HMA0214-48 was tested to see whether it induces red blood cell agglutination. The highest antibody concentration was 667 nM, the antibodies were serially diluted, and a total of 12 concentration points were tested.

As shown in figure 9, it showed that CD47 antibody Hu5F9 could significantly induce red blood cell agglutination when its concentration is 0.9 nM or above. By contrast, the antibody HMA02h14-48 in the present invention did not induce a significant hemagglutination of human red blood cells in vitro at different concentrations from 0.004 to 667 nM.

Example 9 Detection of binding activity of HMA02h14-48 to human red blood cells by FACS

It is known that in the prior art, when therapeutic anti-CD47 antibodies are used clinically, side effects such as anemia often occur. It is generally believed that anti-CD47 antibodies bind to CD47 on the surface of red blood cells, which would in turn cause the phagocytosis of red blood cells by macrophages. This could be another major cause of anemia. In the present invention, flow cytometry was used to detect the binding ability of HMA02h14-48 to human red blood cells to evaluate the risk of antibodies. Specifically, red blood cells from healthy donors were incubated with diluted HMA02h14-48 (the maximum concentration being 667 nM, 8 test concentration points in total) in PBS containing 2%fetal bovine serum at 4℃ for 30 minutes. Then the cells were washed three times and incubated with the secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, Cat: 409304) in PBS containing 2%fetal bovine serum at 4℃ in the dark for 30 minutes. The cells were washed three times with PBS containing 2%fetal bovine serum (FBS) , and then the fluorescence signal was detected by Canto II (BD Biosciences) flow cytometry. According to the average fluorescence intensity (MFI) of the signal, GraphPad was used for fitting a concentration dependent curve, and the EC ₅₀ was calculated.

As shown in figure 10, the maximum mean fluorescence intensity of HMA02h14-48 bound to CD47 on the surface of human red blood cells was lower than that of control antibody Hu5F9. The maximum mean fluorescence intensity and EC ₅₀ thereof was shown in Table 9.

Table 9 Binding activity of antibody HMA02h14-48 to CD47 on the surface of human red blood cells

Example 10 Inhibition of Toledo Tumor Growth by Humanized Antibody HMA02h14-48

Objectives: A Toledo subcutaneous tumor model was established in NOD-Scid mice to study the anti-tumor activity of the antibody of the present invention.

Methods: human diffuse large B-cell lymphoma cells Toledo (

CRL-2631 ^TM) was cultured with RPMI1640 medium containing 10%fetal bovine serum. Tumor cells were suspended in RPMI1640 and implanted into male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd. ) subcutaneously in the right flank at a dose of 1 × 10 ⁷ cells/mouse.

15 days after tumor cell inoculation, mice were randomly divided into 6 groups according to tumor volume, Hu5F9 and HMA02h14-48 antibodies were diluted with PBS respectively, and the mice were administered at a dose of 10 mg/kg according to the schedule shown in Table 10. The negative isotype control antibody (isotype) IgG4P was purchased from Shanghai ChemPartner Co., Ltd.

Table 10. Dosage regimens of Hu5F9 and HMA02h14-48

Note: The grouping day is defined as day 0, and the next day for drug administration is day 1.

The tumor volumes (tumor volume = 0.5 × long diameter × short diameter ²) and body weights of the mice were measured regularly. The changes in tumor volume and body weight were statistically analyzed using student t-test in Excel software, wherein p < 0.05 indicates a significant statistical difference. The tumor regression rate of each antibody treatment group after administration was calculated.

The formula for calculating tumor regression rate in each treatment group is: [ (D ₀ average tumor volume-D _t average tumor volume) /D ₀ average tumor volume] × 100%.

The formula for calculating the relative weight of a mouse is: (weight of the mouse on the day of measurement/weight of the mouse at the time of grouping) × 100%.

Results:

The experimental results are shown in Table 11 and Figure 11.

The tumors in the isotype control antibody group grew well, while in therapeutic antibody treatment groups, the subcutaneous tumor volume gradually reduced compared with the initial volume until completely regressed. Groups with Hu5F9 and HMA02h14-48 antibodies administered at various doses achieved the effect of complete tumor regression (regression rate of 100%) when measured on day 11, compared with the control antibody in the control group, the tumor volume reduction were statistically significant. After the dosing discontinuation, the animals were observed until day 67, and there was still no sign of tumor regrowth. In addition, the animals in groups with HMA02h14-48 administered at various doses were in good status, and there was no significant difference in the body weights of the mice on day 21 compared with that before treatment. The body weight of mice in the group with a high dose of Hu5F9 on day 21 was reduced by about 5%compared with that on day 0, but there was no statistical difference compared with the initial weight (p > 0.05) ; however, there was no weight loss in the low-dose group of Hu5F9, suggesting a possible dose-related effect of Hu5F9 on body weight.

Based on the above-mentioned data, both Hu5F9 and HMA02h14-48 antibody treatments showed extremely significant anti-tumor effects. A single dose of either antibody at 10 mg/kg led to complete regression of the tumor and the duration is longa. Table 11 Effect of Hu5F9 and HMA02h014-48 on Toledo subcutaneously transplanted tumor growth

Note: **is p < 0.01; The numbers in parentheses are tumor regression rates.

Example 11 Inhibition of REC-1 tumor growth by humanized antibody HMA02h14-48

Objectives: a REC-1 subcutaneous tumor model was established in NOD-Scid mice to study the anti-tumor activity of the antibody of the present invention.

Methods: human mantle cell lymphoma cells REC-1 (

CRL-3004 ^TM) was cultured in RPMI1640 medium containing 10%fetal bovine serum. Tumor cells were suspended in RPMI1640 and implanted into male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd. ) subcutaneously in the right flank at a dose of 5 × 10 ⁶ cells/mouse.

11 days after tumor cell inoculation, mice were randomly divided into 5 groups according to tumor volume, Hu5F9 and HMA02h14-48 antibodies were diluted with PBS, and the mice were administered according to the schedule shown in Table 12. The antibody Hu5F9 was prepared by GenScript, and the antibody HMA02h14-48 was prepared according to the method in Example 2. The isotype control antibody (isotype) IgG4p was purchased from Shanghai ChemPartner Co., Ltd.

Table 12. Dosing regimens of Hu5F9 and HMA02h14-48

The tumor volumes (tumor volume = 0.5 × long diameter × short diameter ²) , and body weights of the mice were measured regularly. The tumor inhibition rate and regression rate of the antibody treatment group on day 12 after administration were calculated.

The formula for calculating tumor inhibition rate is as follows: [ (average tumor volume change in the control group-average tumor volume change in the treatment group) /average tumor volume change in the control group] × 100%. The changes in tumor volume and body weight were statistically analyzed using Student t-test in Excel software, wherein p < 0.05 indicates a significant statistical difference.

Results:

The experimental results were shown in Table 13 and Figure 12.

12 days after administration, compared with Isotype group, the tumor growth inhibition rate was 16.7% (p > 0.05) in the group treated with a single dose of Hu5F9 at 3 mg/kg; and the tumor growth inhibition rates were 3.8% (p > 0.05) , 54.7% (p < 0.01) and 107.2% (p < 0.001) , respectively, in the groups with a single dose of HMA02h14-48 at 1 mg/kg, 3 mg/kg and 10 mg/kg. Groups treated with a high dose of HMA02h14-48 antibody achieved complete tumor regression (regression rate of 100%) on day 10. In addition, there was no significant difference in the relative body weight of the mice in different treatment groups.

Taken together, HMA02h14-48 antibody showed dose-dependent effect in REC-1 model, and a single dose of 10 mg/kg led to complete tumor regression.

Table 13 Effect of Hu5F9 and HMA02h14-48 on REC-1 subcutaneously implanted tumor growth

Note: **is p < 0.01; The numbers in parentheses are tumor regression rates.

Pharmaceutical formulation of the present invention

Material and method

Information of the main materials used in the preparation of the formulation.

Table 14 Information of main materials

Detection items and detection methods of formulation stability

Protein concentration (mg/mL)

The protein concentration in the sample was determined by using an ultraviolet spectrophotometer (LUNATIC, Lunatic-16) .

Size exclusion-chromatography (SEC-HPLC)

The method can be used to separate molecules mainly based on the differences in their sizes or hydrodynamic radii. Antibodies can be separated in three main species by the SEC-HPLC method: high molecular weight species (HMMS) , main peak (mainly antibody monomer) , and low molecular weight species (LMMS) . The purity of the antibody can be calculated as the percentage of the main peak area to the sum of all peak areas on the chromatogram. The percentage of antibody monomers and the content of soluble aggregates and splices (fragments) in the formulation can be measured by the SEC-HPLC method . For further description of the SEC-HPLC method, see, e.g., J. Pharm. Scien., 83: 1645-1650, (1994) ; Pharm. Res., 11: 485 (1994) ; J. Pharm. Bio. Anal., 15: 1928 (1997) ; J. Pharm. Bio. Anal., 14: 1133-1140 (1986) .

SEC-HPLC can be carried out using the following parameters:

Chromatographic column: TSK-

G3000SWXL (7.8 × 300 mm, 5 μm)

Mobile phase: 100 mM PB, 100 mM Na ₂SO ₄·10H ₂O, pH 6.7 ± 0.1

Flow rate: 0.7 mL/min

Column temperature: not controlled

Detection wavelength: 280 nm

Sample load: 1 μL sample

Sample injector temperature: 15℃

Run time: 25 min

Loading concentration: 50 mg/mL

Non-reduced capillary gel electrophoresis-sodium dodecyl sulfate (CE-SDS-NR)

The non-reduced CE-SDS method is a method of determining the purity of monoclonal antibodies using a capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, which surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, an electrophoretic separation based on the size or hydrodynamic radius of the molecules can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to determine the purity of denatured intact antibodies. For further description of the CE-SDS method, see, e.g., Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

Briefly, in a centrifugal tube, 100 μg of proteins was added, followed by a sample buffer -sodium dodecyl Sulfate solution (SDS-MW) to the final volume of 95 μL, then 2 μL of the internal standard (10 kDa protein) , and 5 μL of 250 mM iodacetamide (IAM) were added, and the mixture was mixed evenly and incubated at 70℃ for 10 min. Following incubation, the sample was cooled to room temperature and 90 μL was transferred to a PCR tube. CE-SDS separation was carried out on (model PA800 PLUS, manufactured by SCIEX) . The protein migration is detected using an ultraviolet detector to obtain an electrophoresis spectrogram. The purity of the antibody formulation can be calculated as the percentage of the IgG main peak area to the sum of all peak areas.

Imaged capillary isoelectric focusing electrophoresis method (icIEF method)

By this method, a quantitative distribution of charge variants can be provided. The purpose of separating molecules can be realized by icIEF method based on the difference in their charges in a pH gradient (apparent pI value) . In icIEF, the separation column is typically a short capillary (e.g., a silica capillary, 5 cm in length and 100 μm in inner diameter) , the proteins are focused in the capillary column at a high voltage, and the focusing is monitored online in real time by a whole column imaging detection system at 280 nM. One advantage of this technique is that various charge variants of an antibody sample can be simultaneously recorded by the whole column detection system. Generally, in icIEF, the sample is mixed with urea and an icIEF buffer containing methylcellulose, pI molecular weight standards, and ampholytes. Then the absorbance at 280 nm is determined after the sample has been focused for a period of time on an icIEF analyzer such as an iCE280 analyzer (Protein Simple, Santa Clara, CA) equipped with an icIEF column such as a Protion Simple assembled icIEF column to obtain a spectrum of the focused mAb charge variants. In the iCEIF spectrum, protein-related peaks eluted before the main peak (i.e., a principal component) are classified as acidic components, while protein-related peaks eluted after the main peak are classified as basic components. The relative amounts of the principal component, acidic component and basic component can be expressed as a percentage of the total peak area. For further description of icIEF, see, e.g., Salas-Solano O et al., Robustness of icIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study, J Sep Sci. 2012 Nov; 35 (22) : 3124-9. doi: 10.1002/jssc. 201200633. Epub 2012 Oct 15.

In short, icIEF can be performed as follows: the antibody sample was diluted (or desalted) to about 5 mg/mL, and 4 μL of the sample was added to 96 μL of a buffer (the buffer contains 4%ampholytes, 0.35%methylcellulose, 1%PI marker (propidium iodide marker) , 500 mM arginine, and 4 M urea) . After mixing, an imaged capillary isoelectric focusing spectrum analysis was carried out on a Maurice protein characterization analyzer (Protein Simple, Santa Clara, CA) .

Protein Activity (by ELISA)

ELISA (enzyme-linked immunosorbent assay) can be used to test the ability of the antibody in the formulation to bind to its antigen, so as to evaluate the biological activity of the antibody.

Osmolality

The osmolality of the formulation sample was measured on a 3250 osmometer from Advanced Instruments according to the principle of freezing point depression.

pH

The pH of the formulation sample was determined by potentiometry with a glass electrode, the device used is a pH meter S400-B from METTLER TOLEDO, and the pH meter was calibrated with standard solutions (the pH being 4.01, 7.00 and 9.21) prior to measuring the sample.

Example 12. Pharmaceutical formulation stability study 1

The antibody HMA02h14-48 prepared as above in Example 2 was used in the pharmaceutical formulation Examples and was referred to as mAb1 in the pharmaceutical formulation studies.

Various groups of candidate formulation were designed as in Table 16, and the effects of different pH/buffers, antibody concentrations, and stabilizers or surfactant concentrations on the antibody stability were investigated.

Experiment method

1) mAb1 antibody samples (see Table 15 for the sample information, citrate buffer is citric acid -disodium citrate buffer herein) were taken, the corresponding buffer solutions and the stock solution of arginine hydrochloride were added according to the schemes in Table 16, and arginine hydrochloride was adjusted to the target concentration; for E6, the stock solution of citrate buffer was also added and citrate was adjusted to the target concentration; and for E3 and E5, the solution was replaced with the corresponding histidine salt buffer containing arginine hydrochloride and then the antibody was concentrated; and for other prescription samples, the solutions were directly concentrated; 2) the corresponding stock solution of sucrose (as desired) and the stock solution of polysorbate 80 were added; 3) the corresponding buffer solutions were added and various components were adjusted to the target concentrations; and 4) the samples were filtered and sterilized in a biosafety cabinet by using a 0.22 μm disposable sterile filter and sub-packaged (1 mL/vial) ; and the vials were stoppered, capped and labeled.

Testing scheme: stability testing was carried out according to the sampling schedule in Table 17 under the conditions of a high temperature (40℃) and freezing and thawing (-70℃ ± 10℃/5℃ ± 3℃) .

Table 15 mAb1 antibody sample information

Table 16 Candidate formulation information

In Table 16, the citrate buffer is citric acid -disodium citrate buffer, the histidine salt buffer is histidine-histidine hydrochloride buffer.

Table 17 Experimental testing time points and items

X: Protein concentration, SEC-HPLC, CE-SDS-NR, icIEF;

Y: ELISA

Results and analysis

(1) Protein concentration determination results

The protein concentration determination results were shown in Table 18. At T0, the protein concentration of each candidate formulation sample was consistent with the requirements of experimental design and showed no significant difference. After 5 cycles of freezing and thawing at -70℃/5℃, and after being placed at a high temperature of 40℃ for 4 weeks, candidate formulation showed no significant change in terms of the protein concentration compared to T0, demonstrating good stability at the high temperature and good freeze-thaw stability.

Table 18 Testing under the conditions of 40℃ and freezing and thawing: protein concentration determination results

(2) Purity (by SEC-HPLC)

The results for purity determination (by SEC-HPLC) were shown in Tables 19 and 20. At T0, the purity (by SEC-HPLC) of each candidate formulation sample showed no significant difference.

With extended testing time at a high temperature of 40℃, all formulations showed no significant change in terms of the main peak purity (by SEC-HPLC) compared to T0, demonstrating good stability at the high temperature.

The freezing and thawing results indicate that after 5 cycles of freezing and thawing, each prescription sample showed no significant change in terms of the purity (by SEC-HPLC) compared to T0, which showed that the mAb1 antibody in each candidate prescription was freeze-thaw stable.

Table 19 Testing under the condition of 40℃: SEC-HPLC determination results

Table 20 Testing under freezing and thawing conditions: SEC-HPLC determination results

(3) Purity (by CE-SDS-NR)

The results for purity determination (by CE-SDS-NR) were shown in Table 21. The results of this study showed that at T0, the purity (by CE-SDS-NR) of each candidate formulation showed no significant difference. After being placed at 40℃ for 4 weeks, all candidate formulation samples showed no significant change in terms of the monomer purity compared to T0, demonstrating good stability at the high temperature.

After 5 cycles of freezing and thawing, all candidate formulation showed no significant change in terms of the monomer purity, and there was no significant difference among the prescriptions.

Table 21 Testing under the conditions of 40℃ and freezing and thawing: CE-SDS-NR determination results

*:It can be determined according to the trend that the CE-SDS-NR result of the sample E4 at T0 was abnormal and for the result at T0, reference can be made to the determination result obtained after one cycle of freezing and thawing.

(4) Purity (by icIEF)

The results for purity determination (by icIEF) were shown in Tables 22 and 23. At T0, candidate formulation samples showed no significant difference in terms of the icIEF results. After testing under the conditions of 40℃ for 4 weeks, each formulation showed no significant change in terms of the main peak purity compared to T0, demonstrating good stability at the high temperature.

After testing under freezing and thawing conditions for 5 cycles, all formulations showed no significant change in terms of the main peak purity, acidic peak content and basic peak content, demonstrating good freeze-thaw stability.

Table 22 Testing under the condition of 40℃: icIEF determination results

Table 23 Testing under freezing and thawing conditions: icIEF determination results

(5) Relative binding activity of proteins by ELISA

The determination results of the activity were shown in Table 24. At T0, the activity of each candidate formulation sample was within the range of 70%-130%and showed no significant difference. After being placed at a high temperature of 40℃ for 4 weeks and after 5 cycles of freezing and thawing, the activity of each formulation was maintained within the range of 70%-130%and was consistent with the quality requirements compared to T0.

Table 24 Testing under the conditions of 40℃ and freezing and thawing: Results for determination of relative binding activity of proteins by ELISA

To sum up, within the 4-week testing at the high temperature of 40℃ or after testing under freezing and thawing conditions for 5 cycles, the pharmaceutical formulations showed no significant difference in terms of protein concentration, and purity (by SEC-HPLC, CE-SDS-NR and icIEF) and had good stability at the high temperature and good freeze-thaw stability.

Example 13: Pharmaceutical formulation stability study 2

Based on the aforesaid study results, under isotonic conditions, pharmaceutical formulations were designed to further compare and confirm 1) the effects of the ratios of arginine hydrochloride to sucrose, and 2) the effects of the concentrations of polysorbate 80. The aim was to determine the stability performance of the formulation when arginine hydrochloride, sucrose, and polysorbate 80 were used in the boundary amounts.

Experimental method

mAb1 antibody sample (see Table 25 for the sample information, the citrate buffer is citric acid -disodium citrate buffer herein) were taken and the corresponding buffer solution (20 mM citrate buffer, i.e. citric acid -disodium citrate buffer ) was added according to the schemes in Table 26.1) For F1, F2, F7, and F8, the stock solution of arginine hydrochloride and the stock solution of polysorbate 80 were added and finally the buffer solution was added to adjust each component to the target concentration; 2) for F3 and F4, the stock solution of arginine hydrochloride, the stock solution of sucrose and the stock solution of polysorbate 80 were added and the buffer solution was added to adjust each component to the target concentration; 3) for F5, the corresponding stock solution of sucrose and stock solution of polysorbate 80 were added and finally the buffer solution was added to adjust each component to the target concentration; 4) for F6, 20 mM citrate buffer was used to dilute arginine hydrochloride to the target concentration and then the antibody was concentrated, the corresponding stock solution of sucrose and stock solution of polysorbate 80 were added and then the buffer solution was added to adjust each component to the target concentration; and 5) the samples were filtered and sterilized in a biosafety cabinet by using a 0.22 μm disposable sterile filter and sub-packaged (1 mL/vial) ; and the vials were stoppered, capped and labeled.

Testing scheme: stability testing at a high temperature of 40℃ was carried out according to the sampling schedule in Table 27.

Table 25 mAb1 antibody sample information

Table 26 Candidate prescriptions of pharmaceutical formulation stability study 2

Table 27 Experimental testing time points and items

X: Protein concentration, SEC-HPLC, CE-SDS-NR, icIEF;

Y: ELISA

Experimental results

(1) Results for determination of osmolality

The results for determination of the osmolality of each candidate pharmaceutical formulation were shown in Table 28. The osmolality of each candidate formulation sample was isotonic.

Table 28 Osmolality determination results of candidate formulations

Sample number	Osmolality (mOsmol/kg)
F1	311
F2	281
F3	283
F4	282
F5	302
F6	331
F7	283
F8	282

(2) Protein concentration determination results

The determination results of the protein concentration were shown in Table 29. At T0, the protein concentration of each candidate formulation sample was consistent with the requirements of experimental design and the formulations showed no significant difference. After being placed at a high temperature of 40℃ for 4 weeks, each candidate formulation showed no significant change in terms of the protein concentration compared to T0, demonstrating good stability at the high temperature (40℃) .

Table 29 Protein concentration determination results

(3) Purity (by SEC-HPLC)

The results for purity determination (by SEC-HPLC) were shown in Table 30. At T0, the purity (by SEC-HPLC) of each candidate formulation sample showed no significant difference. After testing at a high temperature of 40℃ for 4 weeks, all formulations showed no significant change in terms of the main peak purity (by SEC) compared to T0, demonstrating good stability at the high temperature (40℃) .

Table 30 SEC-HPLC determination results

(4) Purity (by CE-SDS-NR)

The results for purity determination (by CE-SDS-NR) were shown in Table 31. The results of this study showed that at T0, the purity (by CE-SDS-NR) of each candidate formulation showed no significant difference. After being placed at a high temperature of 40℃ for 4 weeks, all candidate formulation samples showed no significant change in terms of the monomer purity compared to T0, demonstrating good stability at the high temperature (40℃) .

Table 31 CE-SDS-NR determination results

(5) Purity (by icIEF)

The results for purity determination (by icIEF) were shown in Table 32. After testing at the condition of a high temperature (40℃) for 4 weeks, each formulation showed no significant change in terms of the main peak purity compared to T0, demonstrating good stability at the high temperature (40℃) .

Table 32 Results for determination by icIEF after testing under the condition of 40℃with three rounds of excipient screenings

(6) Relative binding activity of proteins by ELISA

The determination results of the activity were shown in Table 33. At T0, the activity of each candidate formulation sample was within the range of 70%-130%and showed no significant difference. After being placed at a high temperature of 40℃ for 4 weeks, the activity of each formulation was maintained within the range of 70%-130%compared to T0, and had good stability in terms of the activity and there was no significant difference among the formulations.

Table 33 Results for determination of relative binding activity of proteins by ELISA

To sum up, after testing at a high temperature of 40℃ for 4 weeks, formulations showed no significant difference in terms of protein concentration and purity (by SEC-HPLC, CE-SDS-NR and icIEF) . It was found that different concentration ratios of arginine hydrochloride to sucrose had no significant effects on the protein stability.

Sequences of the present invention:

The examples and embodiments of the present invention are illustrated above. It is to be understood by those skilled in the art that, these disclosures are only for interpretation purpose, and various substitutions, adaptations and modifications would be made within the scope of the present invention. Accordingly, the present invention is not to be interpreted as being limited to the specific embodiments and examples listed herein.

Claims

A stable pharmaceutical formulation, comprising

(i) an anti-CD47 antibody or an antigen-binding fragment thereof;

(ii) a buffer;

(iii) a stabilizer; and

(iv) a surfactant,

with a pH of about 5.0 to about 8.5, and

wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

(1) one to three selected from HCDR1, HCDR2 and HCDR3 of a heavy chain variable region (VH) , wherein VH comprises the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7; and/or

(2) one to three selected from LCDR1, LCDR2 and LCDR3 of a light chain variable region (VL) , wherein the VL comprises the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.
The pharmaceutical formulation according to claim 1, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

HCDR1, HCDR2 and HCDR3 of a VH and

LCDR1, LCDR2 and LCDR3 of a VL, wherein the VH and VL are selected from:

(1) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 2;

(2) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 3, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(3) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 5, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10;

(4) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 6, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 9 or 10; or

(5) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.
The pharmaceutical formulation according to claim 1, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

(1) one to three selected from heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21; and/or

(2) one to three selected from light chain complementary determining region 1 (LCDR1) , LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 16.
The pharmaceutical formulation according to claim 3, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

(1) heavy chain complementary determining region 1 (HCDR1) , HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 13 or 17 or 21; and/or

(2) light chain complementary determining region 1 (LCDR1) , LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 15 or 18 or 22, and the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 16.
The pharmaceutical formulation according to claim 3, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

(1) the HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 17; and/or

(2) the LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 16.
The pharmaceutical formulation according to any one of claims 1-3, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises

(1) a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 6 or 7; and/or

(2) a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 8, 9 or 10.
The pharmaceutical formulation according to claim 6, wherein the VH comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 6 and 7, and the VL comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 8, 9 and 10.
The pharmaceutical formulation according to claim 7, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , and wherein the VH and VL are selected from:

(1) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 1; and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 2;

(2) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 3; and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(3) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 5; and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10;

(4) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 6; and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 9 or 10; or

(5) a VH comprising the amino acid sequence as set forth in SEQ ID NO: 7; and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.
The pharmaceutical formulation according to claim 1, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 8.
The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises a heavy chain constant region, such as a human IgG1 constant region, a human IgG1TM constant region, a human IgG4 constant region or a human IgG4P constant region.
The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises a light chain constant region, such as a human κ light chain constant region.
The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-CD47 antibody is a murine antibody, a chimeric antibody or a humanized antibody.
The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-CD47 antibody or the antigen-binding fragment thereof is a full-length antibody, a single-chain antibody, a single-domain antibody such as a VHH, a Fab, a Fab’, a Fab’-SH, a (Fab’) ₂, a single-chain antibody such as a scFv, a Fv, a dAb (domain antibody) or a bis (multi) -specific antibody.
The pharmaceutical formulation according to any one of the preceding claims, wherein the buffer is a histidine salt buffer or a citrate buffer.
The pharmaceutical formulation according to any one of the preceding claims, wherein the buffer is at a concentration of about 10 mM to about 80 mM, preferably about 20 mM to about 50 mM, such as about 20 mM or about 50 mM.
The pharmaceutical formulation according to any one of the preceding claims, wherein the stabilizer is at least one selected from the group consisting of a sugar, an amino acid, and a polyol.
The pharmaceutical formulation according to any one of the preceding claims, wherein the stabilizer comprises at least one amino acid, such as arginine, preferably arginine hydrochloride.
The pharmaceutical formulation according to any one of the preceding claims, wherein the stabilizer consists of an amino acid and a sugar, preferably arginine hydrochloride and sucrose.
The pharmaceutical formulation according to claims 16-18, wherein the amino acid is at a concentration of about 1 mg/mL to about 100 mg/mL, such as 5 mg/mL to about 40 mg/mL.
The pharmaceutical formulation according to claim 16 or 19, wherein the sugar is at a concentration of about 10 mg/mL to about 100 mg/mL, preferably about 20 mg/mL to about 80 mg/mL.
The pharmaceutical formulation according to any one of claims 16-20, wherein the ratio of mass concentration of the amino acid to the sugar is about 1: 1 to about 1 : 15.
The pharmaceutical formulation according to any one of the preceding claims, wherein the surfactant is selected from polysorbate surfactants, preferably polysorbate-80 or polysorbate-20.
The pharmaceutical formulation according to any one of the preceding claims, wherein the surfactant is at a concentration of about 0.002% (w/v) to about 0.5% (w/v) , pereferably about 0.01% (w/v) to about 0.05% (w/v) , such as about 0.01% (w/v) , about 0.02% (w/v) , or about 0.03% (w/v) , preferably about 0.02% (w/v) .
The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-CD47 antibody is at a concentration of about 10-150 mg/mL, preferably about 40-120 mg/mL, such as about 50 mg/mL, about 80 mg/mL or about 100 mg/mL.
The pharmaceutical formulation according to any one of the preceding claims, wherein the pH is about 5.5 to about 7.0, preferably about 6.0 to about 7.0.
The pharmaceutical formulation according to any one of claims 1-13, comprises:

(i) about 10-150 mg/mL of the anti-CD47 antibody or the antigen-binding fragment thereof;

(ii) about 10-80 mM of the citrate buffering agent or the histidine salt buffering agent;

(iii) about 5-40 mg/mL of arginine hydrochloride and about 0-80 mg/mL of sucrose; and

(iv) about 0.01% (w/v) to about 0.05% (w/v) of polysorbate-80,

with a pH of about 6.0-7.0.
The pharmaceutical formulation according to any one of claims 1-13, comprises:

(i) about 40-120 mg/mL of the anti-CD47 antibody or the antigen-binding fragment thereof;

(ii) about 20-50 mM of the citrate buffer or the histidine salt buffer;

(iii) about 5-40 mg/mL of arginine hydrochloride and about 20-80 mg/mL of sucrose, wherein the ratio of mass concentration of arginine hydrochloride to sucrose is about 1 : 1 to about 1 : 15, and

(iv) about 0.01% (w/v) to about 0.05% (w/v) of polysorbate-80,

with a pH of 6.0-7.0.
The pharmaceutical formulation according to any one of claims 1-13, comprises:

(i) about 40-120 mg/mL of the anti-CD47 antibody or the antigen-binding fragment thereof;

(ii) about 20-50 mM of the citrate buffer;

(iii) about 5-40 mg/mL of arginine hydrochloride, and

(iv) about 0.01% (w/v) to about 0.05% (w/v) of polysorbate-80,

with a pH of about 6.0-7.0.
The pharmaceutical formulation according to any one of the preceding claims, which is in a liquid form or a liquid form reconstituted from a lyophilized form, further comprising a vehicle, such as water.
A lyophilized pharmaceutical formulation, which is prepared, by means of lyophilization, from the pharmaceutical formulation according to any one of the preceding claims.
A reconstituted pharmaceutical formulation, which is prepared by the reconstitution of lyophilized pharmaceutical formulation of claim 30 with a reconstitution medium, wherein the reconstitution medium is at least oneselected from the group consisting of water for injection, bacteriostatic water for injection, a sodium chloride solution, and a glucose solution.
The reconstituted pharmaceutical formulation of claim 31, which further comprises a vehicle, such as water.
The pharmaceutical formulation according to any one of claims 1-29, the lyophilized pharmaceutical formulation of claim 30, or the reconstituted pharmaceutical formulation of claims 31 or 32, for use in the preparation of a drug for treating CD47-related diseases in a subject in need thereof, wherein the CD47-related diseases are preferably cancers, the cancers comprise hematological cancer and solid tumor, such as bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, melanoma, leiomyoma, leiomyosarcomas, glioma, glioblastoma, myeloma, endometrial cancer, renal carcinoma, melanoma, prostate cancer, thyroid carcinoma, cervical cancer, gastric cancer, liver cancer, colon cancer, ovarian cancer, urothelial carcinoma, and so on.
An article of manufacture, comprising a container filled with the pharmaceutical formulation according to any one of claims 1-29, the lyophilized pharmaceutical formulation of claim 30, or the reconstituted pharmaceutical formulation of claims 31 or 32.
A method of treating or preventing CD47-related diseases in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical formulation according to any one of claims 1-29, the lyophilized pharmaceutical formulation of claim 30, or the reconstituted pharmaceutical formulation of claims 31 or 32.
The method according to claim 35, wherein the CD47-related diseases are preferably cancers, for example, the cancers comprise hematological cancer and solid tumor, such as bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, melanoma, leiomyoma, leiomyosarcomas, glioma, glioblastoma, myeloma, endometrial cancer, renal carcinoma, melanoma, prostate cancer, thyroid carcinoma, cervical cancer, gastric cancer, liver cancer, colon cancer, ovarian cancer, urothelial carcinoma, and so on.